Detection of Extracellular Tumor-Associated Nucleic Acid in Blood Plasma or Serum Using Nucleic Acid Amplification Assays

ABSTRACT

This invention relates to detection of specific extracellular nucleic acid in plasma or serum fractions of human or animal blood associated with neoplastic or proliferative disease. Specifically, the invention relates to detection of nucleic acid derived from mutant oncogenes or other tumor-associated DNA, and to those methods of detecting and monitoring extracellular mutant oncogenes or tumor-associated DNA found in the plasma or serum fraction of blood by using rapid DNA extraction followed by nucleic acid amplification with or without enrichment for mutant DNA. In particular, the invention relates to the detection, identification, or monitoring of the existence, progression or clinical status of benign, premalignant, or malignant neoplasms in humans or other animals that contain a mutation that is associated with the neoplasm through detection of the mutated nucleic acid of the neoplasm in plasma or serum fractions. The invention permits the detection of extracellular, tumor-associated nucleic acid in the serum or plasma of humans or other animals recognized as having a neoplastic or proliferative disease or in individuals without any prior history or diagnosis of neoplastic or proliferative disease. The invention provides the ability to detect extracellular nucleic acid derived from genetic sequences known to be associated with neoplasia, such as oncogenes, as well as genetic sequences previously unrecognized as being associated with neoplastic or proliferative disease. The invention thereby provides methods for early identification of colorectal, pancreatic, lung, breast, bladder, ovarian, lymphoma and all other malignancies carrying tumor-related mutations of DNA and methods for monitoring cancer and other neoplastic disorders in humans and other animals.

This application is a continuation of U.S. Ser. No. 09/642,952, filedAug. 21, 2000, which is a continuation of U.S. Ser. No. 08/818,058,filed Mar. 14, 1997, now U.S. Pat. No. 6,156,054, which is acontinuation-in-part of U.S. Provisional Application Ser. No.60/028,180, filed Oct. 15, 1996, and U.S. Provisional Application Ser.No. 60/026,252, filed Sep. 17, 1996, and U.S. Provisional ApplicationSer. No. 60/013,497, filed Mar. 15, 1996, each of which provisionalapplications is now abandoned, the entire disclosure of each of which ishereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for detecting specific extracellularnucleic acid in plasma or serum fractions of human or animal bloodassociated with neoplastic or proliferative disease. Specifically, theinvention relates to detection of nucleic acid derived from mutantoncogenes or other tumor-associated DNA, and to methods of detecting andmonitoring extracellular mutant oncogenes or tumor-associated DNA foundin the plasma or serum fraction of blood by using rapid DNA extractionand nucleic acid amplification. In particular, the invention relates tothe detection, identification, or monitoring of the existence,progression or clinical status of benign, premalignant, or malignantneoplasms in humans or other animals that contain a mutation that isassociated with the neoplasm, through detection of the mutated nucleicacid of the neoplasm in plasma or serum fractions. The invention permitsthe detection of extracellular, tumor-associated nucleic acid in theserum or plasma of humans or other animals recognized as having aneoplastic or proliferative disease or in individuals without any priorhistory or diagnosis of neoplastic or proliferative disease. Theinvention provides the ability to detect extracellular nucleic acidderived from genetic sequences known to be associated with neoplasia,such as oncogenes, as well as genetic sequences previously unrecognizedas being associated with neoplastic or proliferative disease. Theinvention thereby provides methods for early identification ofcolorectal, pancreatic, lung, breast, bladder, ovarian, lymphoma and allother malignancies carrying tumor-related mutations of DNA, and methodsfor monitoring cancer and other neoplastic disorders in humans and otheranimals.

2. Description of the Related Art

Neoplastic disease, including most particularly that collection ofdiseases known as cancer, are a significant part of morbidity andmortality in adults in the developed world, being surpassed only bycardiovascular disease as the primary cause of adult death. Althoughimprovements in cancer treatment have increased survival times fromdiagnosis to death, success rates of cancer treatment are more closelyrelated to early detection of neoplastic disease that enable aggressivetreatment regimes to be instituted before either primary tumor expansionor metastatic growth can ensue.

Oncogenes are normal components of every human and animal cell,responsible for the production of a great number and variety of proteinsthat control cell proliferation, growth regulation, and cell death.Although well over one hundred oncogenes have been described todate-nearly all identified at the deoxyribonucleic acid (DNA) sequencelevel—it is likely that a large number of oncogenes remains to bediscovered.

Genetic mutation as the result of inborn genetic errors or environmentalinsult have long been recognized as playing a causative role in thedevelopment of neoplastic disease. Within the last twenty years,however, the sites of such mutations have been recognized to be withinoncogenes, and mutation of such oncogenes has been found to be anintrinsic and crucial component of premalignant and malignant growth inboth animals and humans. When an oncogene is mutated it alters thegrowth or regulation of the cell through changes in the protein itencodes. If the mutation occurs in a certain region or regions of thegene, or involves a regulatory region of a gene, a growth advantage mayaccrue to a cell having a mutated oncogene. Many malignant tumors orcell lines derived from them have been shown to contain one or moremutated oncogenes, and it is possible that every tumor contains at leastone mutant oncogene.

Mutated oncogenes are therefore markers of malignant or premalignantconditions. It is also known that other, non-oncogenic portions of thegenome may be altered in the neoplastic state. Nucleic acid based assayscan detect both oncogenic and non-oncogenic DNA, whether mutated ornon-mutated. In particular, nucleic acid amplification methods (forexample, the polymerase chain reaction) allow the detection of smallnumbers of mutant molecules among a background of normal ones. Whilealternate means of detecting small numbers of tumor cells (such as flowcytometry) have generally been limited to hematological malignancies(Dressler and Bartow, 1989, Semin. Diag. Pathol. 6: 55-82), nucleic acidamplification assays have proven both sensitive and specific inidentifying malignant cells and for predicting prognosis followingchemotherapy (Fey et al., 1991, Eur. J. Cancer 27: 89-94).

Various nucleic acid amplification strategies for detecting smallnumbers of mutant molecules in solid tumor tissue have been developed,particularly for the ras oncogene (Chen and Viola, 1991, Anal. Biochem.195: 51-56; Kahn et al., 1991, Oncogene 6: 1079-1083; Pellegata et al.,1992, Anticancer Res. 12: 1731-1736; Stork et al., 1991, Oncogene 6:857-862). For example, one sensitive and specific method identifiesmutant ras oncogene DNA on the basis of failure to cleave a restrictionsite at the crucial 12th codon (Kahn et al., 1991, ibid.). Similarprotocols can be applied to detect any mutated region of DNA in aneoplasm, allowing detection of other oncogene DNA or tumor-associatedDNA. Since mutated DNA can be detected not only in the primary cancerbut in both precursor lesions and metastatic sites (Dix et al., 1995,Diagn. Molec. Pathol. 4: 261-265; Oudejans et al., 1991, Int. J. Cancer49: 875-879), nucleic acid amplification assays provide a means ofdetecting and monitoring cancer both early and late in the course ofdisease.

While direct analysis of tumor tissue is frequently difficult orimpossible (such as in instances of occult, unrecognized disease),peripheral blood is easily accessible and amenable to nucleic acidamplification assays such as those mentioned above. Many studies usenucleic acid amplification assays to analyze the peripheral blood ofpatients with cancer in order to detect intracellular DNA extracted fromcirculating cancer cells, including one study which detected theintracellular ras oncogene from circulating pancreatic cancer cells(Tada et al., 1993, Cancer Res. 53: 2472-4). However, it must beemphasized that almost universally these studies attempt to use nucleicacid-based amplification assays to detect extracted intracellular DNAwithin circulating cancer cells. The assay is performed on the cellularfraction of the blood, i.e. the cell pellet or cells within whole blood,and the serum or plasma fraction is ignored or discarded prior toanalysis. Since such an approach requires the presence of metastaticcirculating cancer cells (for non-hematologic tumors), it is of limitedclinical use in patients with early cancers, and it is not useful in thedetection of non-invasive neoplasms or pre-malignant states.

It has not been generally recognized that nucleic acid amplificationassays can detect tumor-associated extracellular mutated DNA, includingoncogene DNA, in the plasma or serum fraction of blood. Furthermore, ithas not been recognized that this can be accomplished in a clinicallyuseful manner, i.e. rapidly within one day, or within less than 8 hours.It is known that small but significant amounts of normal DNA circulatein the blood of healthy people (Fedorov et al., 1986, Bull. Exp. Biol.Med. 102: 1190-2; Leon et al., 1977, Cancer Res. 37: 646-50), and thisamount has been found to increase in cancer states (Shapiro et al.,1983, Cancer 51: 2116-20; Stroun et al., 1989, Oncology 46: 318-322).However, these studies did not employ nucleic acid amplificationmethods, nor did they demonstrate the presence of mutant DNA or specificoncogene DNA in peripheral blood. Thus, the DNAs detected in blood inthese reports were not definitively ascribed to cancer, nor couldclinical utility be realized. In addition, it had been generallypresumed by those with skill in the art that circulating extracellularDNA either does not exist or would be of no clinical utility since itwould be expected to be rapidly digested by plasma DNases. However,inhibitors of DNase appear to be present in the plasma of cancerpatients (Leon et al., 1981, Eur. J. Cancer 17: 533-8). Furthermore,extracellular DNA may exist in proteo-lipid complexes resistant to DNase(Stroun et al., 1987, Eur. J. Cancer Clin. Oncol. 23: 707-12). Inaddition, DNA from tumor cells may be present in the extracellular fluidbecause of secretion or shedding from viable tumor in the form ofproteo-lipid complexes, release of apoptotic bodies from apoptotic tumorcells, or release of free or protein-bound DNA from necrotic or lysedcancer cells. For example, shedding of phospholipid vesicles from tumorcells is well described (Barz et al., 1985, Biochim. Biophys. Acta 814:77-84; Taylor & Blak, 1985, “Shedding of plasma membrane fragments.Neoplastic and developmental importance,” in: Steinberg (ed) The CellSurface in Development and Cancer. Developmental Biology, Plenum Press,New York, pp. 33-57), and similar vesicles have been shown to circulatein the blood of patients with cancer (Carr et al., 1985, Cancer Res. 45:5944-51). Furthermore, DNA has been shown to be present on the cellsurface of tumor cells (Aggarwal et al., 1975, Proc. Natl. Acad. Sci.USA 72: 928-32; Juckett & Rosenberg, 1982, Cancer Res. 42: 3565-73).

Detection of a mutant oncogene in peripheral blood plasma or serum hasbeen the subject of reports in the prior art (see, for example, Sorensonet al., 1994, Cancer Epidemiology, Biomarkers & Prevention 3: 67-71;Vasioukhin et al., 1994, Br. J. Haematol. 86: 774-9; Vasyuldin et al.,1994, “K-ras point mutations in the blood plasma DNA of patients withcolorectal tumors,” in Verna & Shamoo (eds), Biotechnology Today,Ares-Serono Symposia Publications, pp. 141-150). Mutant ras oncogeneshave been demonstrated in plasma or serum using polymerase chainreaction. However, the methods employed by these groups requiredtime-consuming and technically demanding approaches to DNA extractionand are thus of limited clinical utility. Thus, methods that permitmedically useful, rapid, and timely extraction and sensitive detectionof extracellular tumor-associated or extracellular mutated oncogenic DNAare not known in the art.

SUMMARY OF THE INVENTION

This invention provides methods for detecting the presence ofextracellular DNA in blood plasma or serum fractions, said DNA beingassociated with a neoplastic or proliferative disease state in an animalor a human. The invention provides methods for extracting, amplifyingand detecting extracellular DNA associated with a neoplastic orproliferative disease state in an animal or a human and that are usedfor the detection, monitoring, or evaluation of cancer or premalignantconditions.

In a first aspect, the invention provides a method for detectingextracellular tumor-derived or tumor-associated nucleic acid in a plasmaor serum fraction of a blood sample, for diagnosis, detection,monitoring, evaluation or treatment of a neoplastic or proliferativedisease in an animal or a human. The method provided by the inventioncomprises the steps of: first, purifying extracellular nucleic acid fromplasma or serum to prepare a homogeneous preparation of extractednucleic acid; second, specifically amplifying a portion of the extractednucleic acid to provide an amplified nucleic acid fraction comprising anucleic acid that is associated with neoplastic or proliferativedisease; and third, detecting the amplified nucleic acid fragment thatis associated with neoplastic or proliferative disease in the amplifiednucleic acid fraction. In preferred embodiments of this aspect of theinvention, extracted nucleic acid is amplified using an amplificationmethod selected from the group consisting of polymerase chain reaction,ligase chain reaction, branched DNA signal amplification, boomerang DNAamplification, Q-beta replication, transcription-based amplification,isothermal nucleic acid sequence based amplification, self-sustainedsequence replication assay, strand displacement activation, cyclingprobe technology, and combinations or variations thereof. In anotherpreferred embodiment, the nucleic acid is derived from a nucleic acidencoding an oncogene or other tumor-associated DNA.

The invention also provides a method for detecting extracellulartumor-derived or tumor-associated nucleic acid in a plasma or serumfraction of a blood sample, for diagnosis, detection, monitoring,evaluation or treatment of a neoplastic or proliferative disease in ananimal or a human comprising the additional step of digesting theextracted nucleic acid fraction with an enzyme that specifically cleavesnucleic acid in the fraction that is associated with a neoplastic orproliferative disorder, whereby enzymatic cleavage thereof isaccomplished in nucleic acid derived from a wildtype allele of saidnucleic acid that is not associated with a neoplastic or proliferativedisease, but wherein enzymatic cleavage is not accomplished in nucleicacid derived from a mutant or variant allele that is associated with aneoplastic or proliferative disease. Preferably, digestion of theextracted extracellular nucleic acid with an enzyme, preferably anendonuclease, most preferably a restriction enzyme, specifically cleaveswildtype but not mutant DNA in the portion of the sequence between thepositions of the oligonucleotide primers used to amplify the DNA. Thus,wildtype DNA in the sample cannot be amplified after restriction enzymedigestion, whereas mutant DNA can be amplified, and is preferentiallyamplified using the methods of the invention. In a preferred embodiment,the amplification reaction is performed in the presence of athermoresistant or thermostable restriction endonuclease, whichendonuclease specifically cleaves wildtype forms of extracellulartumor-derived or tumor-associated nucleic acid species and therebyinhibits amplification of said species in the amplification reaction. Inanother preferred embodiment, the amplification step of the methods ofthe invention are performed using oligonucleotide primers that produce arestriction endonuclease recognition site in nucleic acid in thefraction that is associated with a neoplastic or proliferative diseasewithin the nucleotide sequence of said nucleic acid fragment, wherebyenzymatic cleavage thereof is accomplished in a nucleic acid fragmentderived from a wildtype allele of said nucleic acid that is notassociated with a neoplastic or proliferative disease, and whereinenzymatic cleavage is not accomplished in a nucleic acid fragmentderived from a mutant or variant allele that is associated with aneoplastic or proliferative disease, and wherein the restrictionendonuclease recognition site is recognized by the thermoresistant orthermostable restriction endonuclease. In other preferred embodiments,endonuclease digestion is performed prior to amplification of theextracted nucleic acid fraction. In a preferred embodiment, the nucleicacid is derived from a nucleic acid encoding an oncogene or othertumor-associated DNA.

In additional preferred embodiments, the invention provides a method fordetecting extracellular tumor-derived or tumor-associated nucleic acidin a plasma or serum fraction of a blood sample, for diagnosis,detection, monitoring, evaluation or treatment of a neoplastic orproliferative disease in an animal or a human comprising the additionalsteps of digesting the amplified nucleic acid fraction with an enzymethat specifically cleaves nucleic acid fragments in the fraction withinthe nucleotide sequence of said nucleic acid fragments, wherebyenzymatic cleavage thereof is accomplished in a nucleic acid fragmentderived from a wildtype allele of said nucleic acid that is notassociated with a neoplastic or proliferative disease, and whereinenzymatic cleavage is not accomplished in a nucleic acid fragmentderived from a mutant or variant allele that is associated with aneoplastic or proliferative disease; then specifically re-amplifying aportion of the amplified, digested nucleic acid that is not cleaved bythe enzyme, to provide a re-amplified nucleic acid fractionsubstantially comprising an undigested nucleic acid that is associatedwith neoplastic or proliferative disease; and detecting the re-amplifiednucleic acid fragment that is associated with neoplastic orproliferative disease in the re-amplified nucleic acid fraction. In thisembodiment of the inventive method, the amplified DNA fragments from theextracellular DNA extracted from plasma or serum is cleaved with anenzyme, preferably a restriction enzyme, that specifically digestsfragments amplified from wildtype alleles of a gene associated with aneoplastic or proliferative disease, and specifically does not cleaveDNA fragments amplified from mutant alleles of a gene wherein themutated allele is associated with a neoplastic or proliferative disease.In a preferred embodiment, the restriction endonuclease is athermoresistant or thermostable endonuclease and digestion is performedsimultaneously with amplification. In another preferred embodiment,digestion is performed with a thermoresistant endonuclease over thecourse of an amplification reaction, whereby wildtype forms of theamplified nucleic acid are specifically cleaved and rendered unamplifiedby the end of the digestion/amplification reaction. In a preferredembodiment, the nucleic acid is derived from a nucleic acid encoding anoncogene or other tumor-associated DNA. In particularly preferredembodiments, an enzyme recognition site is specifically engineered intothe oligonucleotide primers used for amplification to provide an enzymerecognition site in the wildtype allele but not in the mutant allele, asthe result of the nucleotide sequence differences between the wildtypeand mutant alleles. In preferred embodiments, the extracted nucleic acidis amplified using an amplification method selected from the groupconsisting of polymerase chain reaction, ligase chain reaction, branchedDNA signal amplification, boomerang DNA amplification, Q-betareplication, transcription-based amplification, isothermal nucleic acidsequence based amplification, self-sustained sequence replication assay,strand displacement activation, cycling probe technology, andcombinations or variations thereof.

Also provided by the methods of the invention are amplified fragments ofextracellular tumor-associated nucleic acid as detected using themethods of the invention.

Particularly preferred embodiments of the invention compriseamplification of nucleic acid sequences derived from or related to p53,bcl-2 and bcl-2/IgH translocation species.

Preferably the method is provided wherein amplification is achievedusing oligonucleotide primers that specifically amplify a nucleic acidassociated with a neoplastic or proliferative disease, most preferablyan oncogene. In additional preferred embodiments, the amplificationprimers comprise a nested or hemi-nested set of primers as understood inthe art and described herein.

In preferred embodiments of the inventive methods, extracellular nucleicacid is extracted from blood plasma or serum using an extraction methodincluding gelatin extraction; silica, glass bead, or diatom extraction;guanidine- or guanidinium-based extraction; chemical extraction methods;and size-exclusion and anion-exchange chromatographic methods. Inpreferred embodiments, detection of the amplified DNA is performed usinga detection method including gel electrophoresis; immunologicaldetection methods; hybridization using a specific, fluorescent-,radioisotope, antigenic- or chromogenically-labeled probe; Southern blotanalysis; electrochemiluminescence; reverse dot blot detection; andhigh-performance liquid chromatography.

The methods of the invention are provided as diagnostic methods fordetecting tumor-associated extracellular nucleic acid in a human at riskfor developing a neoplastic or proliferative disease (whether the riskis recognized or unrecognized), comprising the steps of purifyingextracellular nucleic acid from a plasma or serum fraction of a bloodsample from the human to prepare a homogeneous preparation of extractednucleic acid; specifically amplifying a portion of the extracted nucleicacid to provide an amplified nucleic acid fraction substantiallycomprising a nucleic acid that is associated with neoplastic orproliferative disease; and detecting the amplified nucleic acid fragmentthat is associated with neoplastic or proliferative disease in theamplified nucleic acid fraction. The detected fragment is thenidentified, e.g., as comprising the wildtype and mutated forms of anoncogene associated with a neoplastic or proliferative disease. In apreferred embodiment, the diagnostic methods of the invention are usedto evaluate response of a human with a neoplastic or proliferativedisease to a treatment regime or modality. In another preferredembodiment, the method is used to evaluate disease progression in ahuman. Additionally, the methods of the invention are preferably used todetermine disease prognosis in a human. In other preferred embodiments,the methods of the invention are used to detect the presence of residualdisease in a human following a course of treatment or after clinicaltumor regression, or to detect actual or imminent clinical relapse.

Also provided as embodiments of the methods of the invention are methodsadditionally comprising the steps of determining the nucleic acidsequence of the nucleic acid fragment of extracellular nucleic acid thatis associated with neoplastic or proliferative disease in the amplifiednucleic acid fraction, wherein the nucleic acid sequence of the nucleicacid fragment comprising a mutated or variant allele of a nucleic acidassociated with a neoplastic or proliferative disease.

In addition to the diagnostic methods noted above, the inventionprovides methods for isolating extracellular tumor-derived ortumor-associated nucleic acid from a fraction of a blood samplecomprising the plasma fraction or the serum fraction of the bloodsample. In these embodiments the method comprises the steps of purifyingextracellular nucleic acid from plasma or serum to prepare a homogeneouspreparation of extracted nucleic acid using a rapid extraction method;specifically amplifying a portion of the extracted nucleic acid toprovide an amplified nucleic acid fraction substantially comprising anucleic acid that is associated with neoplastic or proliferativedisease; and cloning the amplified nucleic acid fragment that isassociated with neoplastic or proliferative disease in the amplifiednucleic acid fraction. Also provided in this aspect of the invention arerecombinant genetic constructs comprising a nucleic acid fragment thatis associated with neoplastic or proliferative disease prepared usingthe methods of the invention. Ribonucleic acid transcribed from therecombinant genetic constructs of the invention are also provided, aswell as protein produced from translation of said RNA, and methods forusing the translated proteins and peptides of the invention as epitopesfor the production of antibodies and vaccines.

In preferred embodiments, the nucleic acid associated with neoplastic orproliferative disease is derived from an oncogene, most preferablywherein the oncogene is ras, p53, bcl-2 or the bcl-2/IgH translocatedgene.

The invention also provides methods for detecting any nucleic acid in asample for which oligonucleotide amplification primers are available.The invention provides a method for detecting a nucleic acid in abiological sample, the method comprising the steps of specificallyamplifying a portion of the nucleic acid in the presence of athermoresistant or thermostable endonuclease to provide an amplifiednucleic acid fraction substantially comprising an amplified nucleic acidfragment; and detecting the amplified nucleic acid fragment. In apreferred embodiment, the nucleic acid is amplified using anamplification method selected from the group consisting of polymerasechain reaction, ligase chain reaction, branched DNA signalamplification, boomerang DNA amplification, Q-beta replication,transcription-based amplification, isothermal nucleic acid sequencebased amplification, self-sustained sequence replication assay, stranddisplacement activation, cycling probe technology, and combinations orvariations thereof. In a preferred embodiment, detection of theamplified DNA is performed using a detection method selected from thegroup consisting of gel electrophoresis, immunological detectionmethods, nucleic acid hybridization using a specific, fluorescent- orchromogenically-labeled probe, Southern blot analysis,electrochemiluminescence, reverse dot blot detection, andhigh-performance liquid chromatography. Nucleic acid from any biologicalsource, including but not limited to eukaryotic, prokaryotic, viral andfungal nucleic acid, can be detected using the inventive method.

It is therefore the object of this invention to detect or infer thepresence of cancerous or precancerous cells from non-hematologic orhematologic malignancies, within a human or animal body havingrecognized neoplastic disease or in those not previously diagnosed, byexamining the plasma or serum fraction of blood for extracellularmutated oncogene DNA or tumor-derived or associated extracellular DNA,using a nucleic acid amplification assay, including but not limited topolymerase chain reaction (PCR), ligase chain reaction, branched DNAsignal amplification assays, isothermal nucleic acid sequence basedamplification (NASBA), other self-sustained sequence replication assays,transcription-based amplification, boomerang DNA amplification,strand-displacement activation, cycling probe technology, orcombinations of such amplification methods, most preferably in thepresence of a restriction endonuclease that specifically cleaveswildtype forms of tumor-derived or associated extracellular nucleicacid.

Another object of this invention is to detect or infer the presence ofcancerous cells anywhere within a human or animal body by examining theplasma or serum fraction of peripheral blood of the organism forextracellular DNA containing mutant oncogene DNA or tumor-associatedDNA, using one or several restriction endonucleases to separatewild-type oncogenes from mutant oncogenes and/or to enrich for mutantDNA, both in organisms known to have cancer and in those not previouslydiagnosed.

Another object of this invention is to rapidly extract extracellular DNAfrom plasma or serum.

An advantageous application of this invention is to identify, eitherquantitatively or qualitatively, mutant oncogenes or tumor-associatedDNA in the blood plasma or serum of humans or animals during orfollowing surgery to remove a premalignant lesion or a cancer, toclassify such patients for their risk of residual cancer or metastasisfollowing the surgery.

Another advantageous application of this invention is to identify,either quantitatively or qualitatively, mutant oncogenes ortumor-associated DNA in the blood plasma or serum of humans or animalswho are receiving cancer therapies, including but not limited tochemotherapy, biotherapy, or radiotherapy, as a guide to whetheradequate therapeutic effect has been achieved or whether additional ormore advanced therapy is required, and to assess prognosis in thesepatients.

Another advantageous application of this invention is to identify,either quantitatively or qualitatively, mutant oncogenes ortumor-associated DNA in the blood plasma or serum of humans or animalswho have completed therapy as an early indicator of relapsed cancer,impending relapse or treatment failure.

Another advantageous application of this invention is to identify,either by detection or inference, the presence of premalignant neoplasmsthrough detection of mutant oncogenes or tumor-associated DNA in theblood of humans or animals when that mutant DNA derives frompremalignant growths such as dysplasias or adenomas, or from other cellsbearing a mutated oncogene. In addition, the invention advantageouslyprovides a panel of several oncogene assays that can distinguishmalignant from premalignant conditions, or assist in medical monitoringto detect transformation of the growth to an outright malignancy, or todetect regression. Furthermore, the invention advantageously provides ameans to define risk of malignancy in a human wherein the risk waspreviously unrecognized.

Thus, the invention provides a method of screening both healthyindividuals and individuals at risk for cancer and premalignantconditions.

Another advantageous application of this invention is to identify,either quantitatively or qualitatively, mutant oncogenes ortumor-associated DNA in the blood plasma or serum of humans or animalseither newly or recently diagnosed with cancer or a premalignantcondition in order to clarify when to initiate therapy, includingadjuvant therapies.

Another advantageous application of this invention is to identify,either quantitatively or qualitatively, more than one mutant oncogene ortumor-associated DNA in the blood plasma or serum of humans or animalsby use of a panel of DNA enrichment methods or by multiplexamplifications of mutant DNAs. Additional, said multiplex amplificationsor collection of individual amplifications of mutant DNAs are providedto identify specific tumor types from the number and kind of oncogenesor other tumor-associated mutated DNAs detected.

Another useful application of this invention is to identify mutantoncogenes or tumor-associated DNA, either singly, multiplexed or using apanel of amplification reactions, in the blood plasma or serum of humansor animals in order to determine specific tumor characteristics for agiven patient, to assist in the development of patient-specifictherapies, or to help place a patient into a particular treatment regimeor to help predict prognosis or tumor behavior.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of the primer construction strategy forcreating diagnostic restriction enzyme digestion sites in PCR amplifiedDNA fragments.

FIG. 2 is a schematic diagram of the serum oncogene detection assay ofthe invention.

FIG. 3 illustrates detection of mutant ras oncogene DNA in serum ofcolorectal cancer patients. An assay of the invention was used toanalyze serum from patients diagnosed with colorectal cancer, and DNAfragments corresponding to mutant K-ras oncogene detected by gelelectrophoresis. In the PCR amplification products of extracellularserum DNA of each of patients A-D was found to a band at the position ofmutated ras oncogene DNA (arrow). Uncut DNA is evident at a highermolecular weight only in the uncut control (dash) indicating completedigestion of all patient samples, while DNA fragments corresponding towildtype ras oncogene DNA runs at a lower molecular weight and isevident in patient D and the negative control (arrowhead). The no-DNAcontrol confirms absence of contamination. Lanes: 1, uncut control; 2,positive control (cell line with mutant ras oncogene); 3, 1:10,000dilution of positive control; 4-7, patients A-D, respectively; 8,negative control (placenta with wild-type K-ras oncogene); 9, no-DNAcontrol; 10, molecular weight markers (φX174 DNA cut with HaeIII).

FIG. 4 shows the results of the assay described in Example 1. No mutantK-ras oncogene DNA was detected in serum of normal donors. The inventiveassay as described in the Example was used to analyze serum from normaldonors, and DNA fragments produced by PCR amplification were detected bygel electrophoresis. Each of the PCR products from normal donor DNAsshowed only the lower molecular weight band indicating only wildtypeK-ras oncogene DNA (arrowhead). The uncut control (dash) and mutatedK-ras oncogene positive control (arrow) are as described in FIG. 3.Lanes: 1 and 13, molecular weight markers (+X174 DNA cut with HaeIII); 2and 14, uncut control; 3, positive control (cell line with mutant K-rasoncogene); 15, 1:10,000 dilution of positive control; 4-12 and 16-23,normal donors.

FIG. 5 shows the bcl-2/IgH transgene is detectable in the serum offollicular lymphoma patients. The assay was used to amplifyextracellular DNA from 4 patients with follicular lymphoma, as describedin Example 2. The transgene is identified in each patient known to havean amplifiable translocation (lanes 3, 5, 6), and not in the patientwithout such a translocation (lane 4). Lanes: 1, molecular weightmarkers (φX174 DNA cut with HaeIII); 2, positive control (cell line withbcl-2/IgH transgene); 3-6, patient serum; 7, no-DNA control.

FIG. 6 shows detection of mutant K-ras oncogene DNA in the plasma of apatient at high risk for development of CRC using the CARD assay. Theassay was used to amplify extracellular DNA from a patient with a strongfamily history of CRC and no clinical signs or symptoms of disease ongross physical examination, as described in Example 1. The mutant K-rasoncogene is indicated (arrow). Lanes: 1, molecular weight markers (φX174DNA cut with HaeIII); 2, uncut control; 3, positive control (cell linewith mutant K-ras oncogene); 4, positive control, diluted 1:100,000; and6, negative plasma; 7, patient sample; 10, negative control (placentalDNA), size of this fragment denoted by a dash to the left of the gelpicture; 11, no-DNA negative control.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods for detecting or inferring the presenceof cancerous or precancerous cells in a human or animal. The methods ofthe invention comprise means of extracting extracellular DNA from bloodplasma or serum in a first step, separating mutated DNA fromnon-mutated, normal DNA by way of a discriminating restrictionendonuclease digestion in a second step, and selectively amplifying anddetecting the DNA in a third step, wherein amplification and detectioncan be performed either qualitatively or quantitatively. The second stepmay be combined with the third step using a thermostable restrictionendonuclease, whereby amplification and digestion/selection areperformed in a single step; this embodiment of the reaction beingdesignated herein as combined amplification and restriction digestion(CARD) assay. This specification describes several methods that can beemployed for the first step (rapid extraction of mutant DNA from plasmaor serum). Similarly, the invention can amplify extracted mutant DNA inthe third step by using any of several methods of nucleic acidamplification and their variations, including but not limited topolymerase chain reaction, ligase chain reaction, branched DNA signalamplification, isothermal nucleic acid sequence based amplification(NASBA), other self-sustained sequence replication assays,transcription-based amplification, boomerang DNA amplification,strand-displacement activation, cycling probe technology, andcombinations of such amplification methods. Specific and detaileddescriptions of several step one methods (rapid extraction of DNA fromserum or plasma) and of several step three methods (nucleic acidamplification of mutant DNA) are given below as a description of theinvention. However, it is emphasized that with this invention any of thedescribed rapid DNA extraction steps may be used with any nucleic acidamplification assay that differentiates mutant DNA or amplifies DNA toachieve the objectives specified above.

Moreover, the assays and methods of the invention can be performedqualitatively, whereby the amount of the nucleic acid product producedis at least sufficient for efficient detection of the product, orquantitatively, whereby the amount of the nucleic acid product producedis measured with reference to a standard useful in determining thesignificance of the amount of produced nucleic acid (for example,wherein the amount of nucleic acid product is related to a disease stateor risk of developing a disease state).

Specifically, the invention provides methods for detecting nucleic acidin plasma or serum of a human or animal wherein the nucleic acid isassociated with the existence of pre-malignant cells or tissues in thehuman or animal, thereby providing a sensitive diagnostic means forearly detection of neoplasia.

A General Overview of the Inventive Methods

In the practice of the invention blood is drawn by standard methods intoa collection tube, preferably comprising siliconized glass, eitherwithout anticoagulant for preparation of serum, or with EDTA, sodiumcitrate, heparin, or similar anticoagulants, most preferably EDTA, forpreparation of plasma. The preferred method, although not absolutelyrequired, is that plasma or serum be fractionated from whole blood.First, this reduces the burden of extraneous intracellular DNA beingextracted from non-malignant cells which might reduce the sensitivity ofthe amplification assay or interfere with the amplification assaythrough release of inhibitors such as porphyrins and hematin. Second,this prevents confounding variables introduced by intracellular DNAderived from circulating cancer cells, for example on interpretation ofquantitative amplification studies. Plasma or serum may be fractionatedfrom whole blood by centrifugation, preferably gentle centrifugation at300-800×g for 5-10 minutes, or fractionated by other standard methods.However, high-speed centrifugation is avoided, as subjecting blood tosuch treatment may deplete the plasma or serum fraction of extracellularDNA. Since heparin may interfere with PCR, use of heparinized blood mayrequire pretreatment with heparinase. Thus, EDTA is the preferredanticoagulant for blood specimens in which PCR amplification is planned.Either freshly-collected blood plasma or serum, or frozen (stored) andsubsequently thawed plasma or serum can be used in the methods of theinvention. Stored plasma or serum should be kept at −20° C. to −70° C.,and freshly-collected plasma or serum kept refrigerated or maintained onice until use.

STEP ONE: Rapid Extraction of Extracellular DNA from Plasma or Serum

I. Gelatin Extraction Method:

In a preferred embodiment, DNA is co-precipitated from plasma or serumwith gelatin by a method modified from that of Fournie et al. (1986,Anal. Biochem. 158: 250-256). A stock 5% (w/v) gelatin solution isprepared by mixing 1 gram gelatin (G8-500, Fisher, Pittsburgh, Pa.) with20 mLs sterile, double-distilled water, autoclaving for 30 minutes, andfiltering through a 0.2 micron filter. The resultant solution issequentially frozen in a dry ice/ethanol bath and thawed at roomtemperature for a total of five cycles. A working 0.3% gelatin solutionis prepared by heating the stock solution to 60° C. and mixing 600 μL of5% gelatin with 25 μL of 1 M Tris-HCl (pH 8.0) and 9.4 mLs of sterile,double-distilled water.

Plasma or serum (160 μL) is mixed with 12.8 μL of 0.5 M EDTA and 467 μL,of sterile, double-distilled water, then emulsified for 3 minutes with320 μL of phenol or phenol:chloroform:isoamyl alcohol (25:24:1 ratio).The solution is centrifuged at 14,000×g for 10 minutes, and 570 μL ofthe aqueous layer is removed to a clean tube. DNA is precipitated byaddition of 142 μL of the 0.3% gelatin working solution and of 500 μL ofcold absolute ethanol, followed by incubation at −20° C. for 1-2 hours.Extracellular DNA may be precipitated within less than 1 h of incubationat −20° C., and a very short incubation may be preferable in somecircumstances. Alternatively, longer incubation at −20° C. for 1-2 hoursinsures the precipitation of most DNA. The sample is centrifuged at14,000×g at 4-6° C. for 15 minutes, washed once with cold 70% ethanol,and dried in a 60° C. heat block for 10 minutes. DNA is then recoveredby the addition of 35 to 70 μL of sterile, double-distilled waterpreheated to 60° C. Thirty-five μL of the resuspended DNA is used ineither step two or step three.

II. Glass Bead, Silica Particle, or Diatom Extraction Method.

As an alternative rapid method of extracting extracellular DNA fromplasma or serum, glass beads, silica particles, or diatoms may be used,as in the method or adaptation of Boom et al. (Boom et al., 1991, J.Clin. Microbiol. 29: 1804-1811; Boom et al., 1989, J. Clin. Microbiol.28: 495-503). Size fractionated silica particles are prepared bysuspending 60 grams of silicon dioxide (SiO₂, Sigma Chemical Co., St.Louis, Mo.) in 500 mLs of demineralized sterile double-distilled water.The suspension is then settled for 24 hours at room temperature.Four-hundred thirty (430) mLs of supernatant is removed by suction andthe particles are resuspended in demineralized, sterile double-distilledwater added to a final volume of 500 mLs. After an additional 5 hours ofsettlement, 440 mLs of the supernatant is removed by suction, and 600 μLof HCl (32% wt/vol) is added to adjust the suspension to a pH2. Thesuspension is aliquotted and stored in the dark.

Lysis buffer is prepared by dissolving 120 grams of guanidinethiocyanate (GuSCN, Fluka Chemical, Buchs, Switzerland) into 100 mLs of0.1 M Tris hydrochloride (Tris-HCl) (pH 6.4), and 22 mLs of 0.2 M EDTA,adjusted to pH 8.0 with NaOH, and 2.6 grams of Triton X-100 (PackardInstrument Co., Downers Grove, Ill.). The solution is then homogenized.

Washing buffer is prepared by dissolving 120 grams of guanidinethiocyanate (GuSCN) into 100 mLs of 0.1 M Tris-HCl (pH 6.4).

Fifty μL of plasma or serum are mixed with 40 μL of silica suspensionprepared as above, and with 900 μL of lysis buffer, prepared as above,using an Eppendorf 5432 mixer over 10 minutes at room temperature. Themixture is then centrifuged at 12,000×g for one minute and thesupernatant aspirated and discarded. The silica-DNA pellet is thenwashed twice with 450 μL of washing buffer, prepared as above. Thepellet is then washed twice with one mL of 70% (vol/vol) ethanol. Thepellet is then given a final wash with one mL of acetone and dried on aheat block at 56 degrees centigrade for ten minutes. The sample iseluted for ten minutes at 56 degrees centigrade with a TE bufferconsisting of 10 mM Tris-HCl, one mM EDTA (pH 8.0) with or withoutProteinase K (100 ng/ml) as described by Boom et al. Following elution,the sample is then centrifuged at 12,000×g for three minutes, and theDNA-containing supernatant recovered. The DNA extract is now used inamplification. (Boom et al., 1991, ibid.; Boom et al., 1989, ibid.;Cheung et al., 1994, J. Clin. Microbiol. 32: 2593-2597).

III. Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction Method.

As an alternative method, extracellular DNA may be extracted from plasmaor serum in step one using variations of the acid guanidiniumthiocyanate-phenol-chloroform extraction method. For example,extracellular DNA may be extracted from plasma or serum using TRIreagent, a monophase guanidine-thiocyanate-phenol solution, as describedby Chomczynski (1993, Biotechniques 15: 532-534). One mL of plasma orserum is processed using 5-10 mLs of TRI Reagent™ (TRI Reagent,Molecular Research Center, Cincinnati, Ohio, Trisolv™, BioTecxLaboratories, Houston, Tex., TRIzol™, GIBCO BRL/Life Technologies,Gaithersburg, Md., ISOGEN™, Nippon Gene, Toyama, Japan, RNA Stat™ 60,Tel-test, Friendsword, Tex.) according to manufacturer's directions. DNAis precipitated from the interphase with ethanol.

IV. Additional Nucleic Acid Extraction Methods

Alternate means of purification which may be used to obtain DNA fromserum or plasma, including selective retention on a size exclusioncolumn or similar matrix, salting-out method, and other guanidiniumthiocyanate extraction methods known in the art.

Combined Amplification and Restriction Digestion (CARD)

The invention provides a particularly preferred embodiment comprising acombined amplification and restriction digestion step, termed CARDassay. This method allows the simultaneous performance of enrichment formutant DNA (Invention Step two, described below) with amplification(invention Step three, described below), significantly shorteninganalysis time and reducing reagent consumption. The method relies uponthe use of a thermoresistant or thermostable restriction endonucleasewhich is able to withstand elevated temperatures (>50° C.) for aprolonged period of time (>5-10 minutes). Thermostable restrictionenzymes generally have reaction conditions similar to those ofthermostable DNA polymerase, so that both enzymes may functionsimultaneously in the same reaction container. The only criterion foruse of the CARD method is that wild-type oncogene DNA carry athermostable restriction enzyme recognition site that is altered inmutant oncogene DNA. If such a site does not exist naturally,oligonucleotide primers may be designed to flank the site of mutationand create a restriction site by altering one or more bases (see FIG.1). Thus, this method has broad application to the rapid selection ofmutant oncogene molecules from a mixture or background of non-mutantoncogene molecules. Indeed, this method may be applied to othersettings, not limited to oncogene DNA detection, in which one form orsequence of DNA is to be selected—on the basis of the presence orabsence of a restriction enzyme site—from another form or sequence ofDNA.

The preferred embodiment of CARD is performed as follows:

DNA is prepared using any of the means described in invention step 1. Amixture of 35 μL of plasma or serum DNA, 50 mM potassium chloride, 10 mMTris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mM magnesium chloride, 200micromolar each DATP, dCTP, dGTP, dTTP, 15 picomole each oligonucleotide(Primers 1 and 2)(the precise amount of each oligonucleotide primer mayvary empirically from one target DNA, to another), 4 units thermostablerestriction endonuclease (the precise amount of each restriction enzymemay vary depending on its degree of thermostability, with more beingneeded for relatively labile enzymes), and 1 unit Taq polymerase(Promega, Madison, Wis.) is prepared in a volume of 50 μL.

In the preferred embodiment, the polymerase chain reaction mixture isincubated at 94° C. for 7 seconds, then at 55-60° C. (depending on thedegree of thermostability of the restriction enzyme and annealingtemperature of the oligonucleotide primers) for 3 minutes, then at 94°C. for 6 seconds, again annealed, extended and digested at 55-60° C. for3 minutes, then incubated at 94° C. for 5 seconds, and so on, decreasingthe length of 94° C. denaturation by one second each cycle until after 6rounds the denaturation lasts only 1 second. Thereafter, cycles with onesecond denaturation steps and 3 minute extension and digestion steps areperformed until a total of 40 is reached. After cycle 10, the reactionsare paused at 60° C. and an additional 10 units of restriction enzymeare added to each tube.

At the completion of the temperature cycling, twenty-five μL of thepolymerase chain reaction (PCR) mixture is then removed to a new tubeand mixed with restriction enzyme reaction buffer and 10 units of thechosen restriction enzyme in a volume of 30 μL, then incubated at theappropriate temperature for reaction to occur for 90 minutes. A secondaliquot is added and the reaction continued for 90 minutes more prior toproceeding to any method of detection specified in invention step three.Alternatively, at the completion of temperature cycling, 10 U of thechosen restriction enzyme are added directly to the cycling reactiontube and this mixture incubated at the appropriate temperature for 1-2 hprior to commencement of the detection step.

The CARD amplification method is also applicable to detecting anynucleic acid in any biological or other sample, wherein amplificationprimers for the nucleic acid of interest are known or may be derived,and in which a restriction enzyme digestion site recognized by athermoresistant or thermostable restriction endonuclease is present orcan be created using the methods of the invention. The use of the CARDmethod of the invention is exemplified but not limited to detection ofextracellular tumor-derived or tumor-associated nucleic acid herein.

STEP TWO: Enrichment for Mutant DNA

Following extraction of extracellular DNA from plasma or serum in stepone, the DNA is amplified using a nucleic acid amplification assay. Oneor more of several amplification assays may be used, includingpolymerase chain reaction, ligase chain reaction, branched DNA signalamplification, isothermal nucleic acid sequence based amplification(NASBA), other self-sustained sequence replication assays,transcription-based amplification, boomerang DNA amplification,strand-displacement activation, cycling probe technology, orcombinations of amplification methods such as polymerase chain reactioncombined with ligase detection reactions. The sensitivity of someamplification assays may be increased by invention step two, which is anoptional step, whereby mutant DNA is enriched through the use of arestriction enzyme, as adapted from the method of Kahn et al. (Kahn etal., 1991, ibid.).

A restriction endonuclease is chosen to examine one portion of a knownoncogene or tumor-associated DNA for mutations. Restrictionendonucleases are naturally occurring enzymes with the ability torecognize a particular arrangement of nucleotide bases and, withabsolute specificity, to cleave double stranded DNA at or near the siteof recognition. Oncogenes such as p53, p16, BRCA1 and ras exhibit anumber of alterations in their DNA sequence that can be identified onthe basis of altered restriction enzyme recognition and cleavage. Thesecond step of the invention uses cleavage of normal, non-mutatedoncogene DNA, by a restriction endonuclease chosen to span one or moreof the nucleotides known to be mutated with some frequency in cancersand their precursors. DNA can then be amplified by any of severalmethods including but not limited to the polymerase chain reaction, theligase chain reaction, self-sustaining sequence replication and others.Since wild-type DNA has been selectively cleaved by restrictionendonuclease digestion, and cleavage prevents DNA amplification, mutantoncogene DNA is relatively enriched following the amplification stage.This cycle of cleavage and amplification may be repeated to furtherenrich the test sample for mutant DNA.

If no restriction enzyme recognition site can be located from anexamination of the known sequence of the oncogene under study, such asite may be created by the introduction of a new base into the sequenceduring a preliminary round of DNA amplification. This method isillustrated in the Example provided below.

In the preferred embodiment, if no restriction enzyme site exists, apreliminary round of DNA amplification is performed as follows. A pairof oligonucleotide primers, each 20-30 nucleotides long, is manufacturedto be complementary to the oncogene being examined (see FIG. 1). One ofthe primers (Primer 1) is designed to lie immediately adjacent to thelocation where mutation occurs in neoplasia. Restriction enzyme sitesare introduced into each of the primers by changing one or twonucleotides as necessary. Primer 1 is altered so that only non-mutated,wild-type DNA is cleaved. Primer 2 is altered to introduce a siterecognized by the same restriction enzyme, which serves as an internalcontrol for digestion.

Primers used in CARD assay or in invention steps two and three should bebased on the specific tumor-derived or associated DNA of interest whichcharacterizes the tumor. Tumor-derived or associated DNA includes but isnot limited to:

-   -   DNA related to mutated oncogenes or other mutated DNA, a partial        list of which includes H-ras, K-ras, A-ras, c-myc, her-2/neu,        bcr-abl, fms, src, fos, sis, jun, bcl-2, bcl-2/IgH, or VHL (Von        Hippel-Lindau gene)    -   DNA related to tumor suppressor genes, a partial list of which        includes p53, RB, MCC, APC, DCC, NF1, WT1.    -   DNA related to tumor-associated protein which is found elevated        in certain cancers, a partial list of which includes        alpha-fetoprotein (AFP), carcinoembryonic antigen. (CEA),        TAG-72, CA 19-9, CA-125, prostate specific antigen (PSA),        epidermal growth factor receptor, and epidermal growth factor    -   DNA related to tumor-derived protein not normally found        circulating in blood, a partial list of which includes        tyrosinase DNA, keratin 19 DNA    -   DNA related to tumor-specific antigens, such as MAGE 1, MAGE 2,        MAGE 3, MAGE 4 and MAGE 4    -   For example, for mutant K-ras oncogene DNA, oligonucleotide        K-ras primers can consist of:

K-ras primer 1 (SEQ ID. No.:1) 5′-ACTGAATATAAACTTGTGGTAGTTGGACCT-3′K-ras primer 2 (SEQ ID. No.:2) 5′-TCAAAGAATGGTCCTGGACC-3′.

The oligonucleotide K-ras primer 1 is immediately upstream of codon 12,and modified at the 28th base (G>C) to create an artificial restrictionenzyme site (BstNI) The oligonucleotide K-ras primer 2 is modified atthe 17th nucleotide (C>G) to create an artificial BstNI site to serve asan internal control for completion of digestion. The amplified mutantK-ras product is of 142 base pair length.

In another example, oligonucleotide p53 primers specific for mutantalleles of the p53 oncogene are shown below in Tables I and II.Specifically, different primers may be utilized in methods of theinvention comprising two amplification steps, allowing for “nesting” or“hemi-nesting” of the amplification products to provide greaterspecificity and decrease the amount of analysis required to detect theamplified product. An example of sets of hemi-nested primers are shownin Tables II and III for mutant p53 oncogene DNA.

In another example, the bcl-2 oncogene DNA, oligonucleotide bcl-2primers can consist of:

MBR 5′-TTAGAGAGTTGCTTTACGTG-3′ (SEQ ID No.:3) J_(H)CON5′-ACCTGAGGAGACGGTGACC-3′ (SEQ ID No.:4) MER-int5′-GCCTGTTTCAACACAGACC-3′. (SEQ ID No.:5)

In a preferred embodiment, polymerase chain reaction is performed as atwo part amplification, in which enrichment of mutant DNA with therestriction enzyme is performed following the first amplification.However, as an alternative to polymerase chain reaction otheramplification methods or their variants may be used, as noted herein.

In a preferred embodiment, a polymerase chain reaction mixtureconsisting of 35 μL of DNA from serum or plasma, 50 mM potassiumchloride, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mM magnesiumchloride, 200 micromolar each dATP, dCTP, dGTP, dTTP, 0.5 picomole eacholigonucleotide (Primers 1 and 2) (the precise amount of eacholigonucleotide primer may vary empirically from one target DNA toanother), and 1 unit Taq polymerase (Promega, Madison, WI) is preparedin a volume of 50 μL.

In a preferred embodiment, the polymerase chain reaction mixture iscycled 15-20 times at 94° C. for 48 seconds, 56° C. for 90 seconds, and72° C. for 155 seconds in an automated thermocycler (Ericomp Deltacycleror similar), as adapted from Kahn (1991, ibid,), prior to restrictionenzyme enrichment of DNA.

Ten μL of the polymerase chain reaction (PCR) mixture is then removed toa new tube and mixed with restriction enzyme reaction buffer and 10units of the chosen restriction enzyme in a volume of 20 μL, thenincubated at the appropriate temperature for reaction to occur for 90minutes. A second aliquot of enzyme is added and the reaction continuedfor 90 minutes more prior to proceeding to step three.

STEP THREE: Nucleic Acid Amplification and Detection

Extracellular DNA which has been extracted from plasma or serum duringstep one, is amplified by a nucleic acid amplification assay utilizedfor detection of low numbers of DNA molecules. Applicable assays includepolymerase chain reaction (PCR), ligase chain reaction, branched DNAsignal amplification, isothermal nucleic acid sequence basedamplification (NASBA), other self-sustained sequence replication assays,transcription-based amplification, Q-beta replication, boomerang DNAamplification, strand-displacement activation, cycling probe technology,and combinations of such amplification methods.

Primers used in the amplification assay should be based on the specifictumor-derived or associated DNA or mutant oncogene DNA of interest whichcharacterizes the tumor, as has been previously described andcharacterized herein (see step two).

I. Polymerase Chain Reaction Amplification:

Amplification reaction specifics using CARD assay are as describedabove, Otherwise, 10 μL of the digested PCR mixture from step two isremoved to a new tube and constituents for another PCR reaction areadded in a volume of 50 μL. All constituents are identical to those instep two except that 15-fold more of each oligonucleotide primer isused. The same cycling conditions are employed for 35 cycles.Alternatively, if invention step two is omitted, an amplificationreaction is prepared using 35 μL of plasma or serum in a final volume of50 μL, with remaining constituents as for the preferred version of stepthree, with the preferred PCR amplification performed in an automatedthermocycler for 15-30 cycles at 94° C. for 48 seconds, 56° C. for 90seconds and 72° C. for 155 seconds per cycle (parameters may be variedand are advantageously optimized for each primer pair).

Following completion of thermocycling, twenty-five μL of this PCRreaction are removed to a new tube, and constituents are added for asecond restriction digestion with the same enzyme. Seventeen units ofenzyme are added in a final volume of 35 μL, with all other constituentsas in the first digestion. The reaction is performed for 60 minutes,followed by addition of 10 additional units of restriction enzyme anddigestion for an additional 60 minutes. The amplified PCR product isthen detected as described herein (see Detection).

If a restriction enzyme site is already present at the point of oncogenemutation, Primer 1 need not contain any mismatches with the knownoncogene sequence and may be placed any convenient distance from thepoint mutation site under examination. Primer 2, however, should stillbe constructed to contain a restriction site cleavable by the sameenzyme to serve as an internal control. In all instances, the PCRproducts should be no larger than 150-200 base pairs, any sequencechange introduced into Primer 1 should be as far from the three primeend as possible, and the sequence change in Primer 2 should create asite that cleaves approximately 10 base pairs from its five prime end.For further clarification, see Example 1 in which a preferred embodimentis used for detection of extracellular mutant K-ras oncogene DNA inplasma or serum.

Other variations of polymerase chain reaction, including quantitativePCR, for example as adapted to the method described by Wang et al.(1989, Proc. Natl. Acad. Sci. USA 86: 9717-9721) or by Karet et al.(1994, Anal. Biochem. 220: 384-390), may alternatively be used.

II. Ligase Chain Reaction Amplification:

Other methods of DNA amplification including ligase chain reaction, andothers as described herein that specifically create new DNA can beemployed with the same effect. The ligase chain reaction (LCR), whichuses a thermostable ligase enzyme to create new double-stranded DNAfragments out of 4 closely opposed oligonucleotides, can be used eitherqualitatively or quantitatively to detect mutant oncogenes in blood asfollows. Oligonucleotides are selected to lie directly upon the oncogenemutation site of interest. The 2 oligonucleotides that are complementaryto the mutation site are manufactured to contain the mutant nucleotidesonly at their three prime ends, thus excluding hybridization to thenon-mutated, wild-type oncogene. If a restriction site exists around thenucleotide(s) of interest, it may be used for restriction digestion toselectively cleave the wild-type molecules. Alternatively, if norestriction site exists one can be created by the introduction ofsequence changes in the oligonucleotides. Finally, if adequatethermodynamic discrimination can be made between mutant and wild-typesequences by the hybridizing oligonucleotides (allele specifichybridization or amplification), no restriction digestions need beperformed and detection of mutant oncogene DNA may proceed directly fromthe DNA harvesting step.

An example of the use of LCR in detection of oncogene DNA in plasma orserum, an assay for K-ras DNA mutated at codon 12 is illustrated.Following extraction of serum or plasma DNA as in step 1, a ligase chainreaction mixture consisting of 35 μL of DNA from serum or plasma, 20 mMpotassium chloride, 20 mM Tris-HCl (pH 7.5), 0.1% NP-40, 10 mM magnesiumchloride, 0.1 mM rATP, 1 mM dithiothreitol, 10 nanograms each of primersLCR1 (5′-ATTACTTGTGGTAGTTGGAGCTGA/T/C-3′; SEQ ID No.:6), where the lastposition is a mixture of three nucleotides A/T/C), LCR2(5′-TGGCGTAGGCAAGAGTGC-3′; SEQ ID No.:7), LCR3(5′-GCACTCTTGCCTACGCCAA/G/T-3′ (SEQ ID No.:8), where the last positionis a mixture of three nucleotides A/G/T), LCR4(5′-CAAGCTCCAACTACCACAAGTAAT-3′; SEQ ID No.:9), and 1 unit Pfu DNAligase (Stratagene, La Jolla, Calif.) is prepared in a volume of 50 μL,The ligase chain reaction mixture is incubated at 92° C. for 4 minutes,followed by 60° C. for 3 minutes, then is cycled 20-25 times at 92° C.for 20 seconds and 60° C. for 20 seconds in an automated thermocycler(Ericomp Deltacycler or similar). This reaction mixture is then used inone or more detection assays as described in step three. This ligationchain reaction depends upon the ability of the ligase enzyme to join twoDNA primers only if they match the target or template DNA (in this case,DNA extracted from serum or plasma) exactly, particularly at the threeprime ends. The mixture of nucleotides at the three prime ends of LCR1and LCR3 will recognize any mutant at the second position of the twelfthcodon, and will effectively amplify it. By contrast, the wild typesequence will not hybridize effectively with these primers, ligationwill not occur, and there will be no amplification of wild type DNA.

III. Alternative Methods of Nucleic Acid Amplification:

An alternative method of either qualitative or quantitativeamplification of nucleic acid which may be used in step three isbranched DNA signal amplification, for example as adapted to the methoddescribed by Urdea et al. (1993, AIDS 7: S11-14; 1991, Nucleic AcidsRes. Symp. Ser. 24: 197-200), modified as follows. Plasma or serum aresubjected to centrifugation at reduced speeds, as previously described,and extracellular DNA extracted as described herein in Step one above.Extracellular DNA is then applied directly to microwells and detectionperformed essentially as described, using target probes specific for thetumor-associated DNA of interest, whereby chemiluminescence is detectedin amounts proportional to the amount of tumor-associated DNA present inthe sample.

An alternative method of either qualitative or quantitativeamplification of nucleic acid which may be used in step three isisothermal nucleic acid sequence based amplification (NASBA), forexample as adapted to the method described by Kievits et al. (1991, J.Virol. Methods 35: 273-286) or by Vandamme et al. (1995, J. Virol.Methods 52: 121-132).

Alternative methods of either qualitative or quantitative amplificationof nucleic acids which may be used in step three include Q-betareplication, other self-sustained sequence replication assays,transcription-based amplification, boomerang DNA amplification,strand-displacement activation, cycling probe technology, andcombinations of amplification methods such as polymerase chain reactioncombined with ligase detection reactions.

Following completion of amplification, the product is detected asdescribed below.

IV. Detection of Amplified Product

There are numerous methods to detect amplified DNA, any of which may beused for detection of amplified product in step three.

In one method, amplified DNA product is detected in step three using gelelectrophoresis. In the preferred embodiment, 25 μL of the seconddigestion product is electrophoresed through a 3% agarose gel in 1×TBEat 75 VDC for approximately 2 hours before staining with ethidiumbromide. Mutant DNA is evident on the gel as a single band of length(PCR product length minus cleaved portion of Primer 2); failure ofdigestion is evident by a band the size of the full-length PCR product,while wild-type, non-mutated DNA is generally not evident but maysometimes be seen as a band at length (PCR product length minus cleavedportion of Primer 2 minus cleaved portion of Primer 1). As analternative to ethidium bromide, the amplified product can betransferred from the gel to a membrane by blotting techniques such asSouthern blot analysis to be detected with a labeled probe.

As an alternative means of detection of the mutant oncogene signal, anytype of hybridization reaction or other method that separatesdifferent-sized PCR products may be employed. For example, anoligonucleotide complementary to the central portion of the PCR productmay be bound to a matrix, and a separate oligonucleotide complementaryto the five prime end of the PCR product, labeled with a fluorescent orchromogenic tag, can be used as a detector. With this format, only PCR,products containing the uncleaved five prime end will hybridize andyield a signal. This approach lends itself to automation and toquantitation, since the fluorescent signal can be cumulated.Additionally, a fluorescent or other tag can be placed on Primer 1 priorto the thermocycling reaction and, with proper adjustment of cyclingparameters, the intensity and thus quantity of mutant oncogene can beread directly following the second round of restriction digestion, as inthe Taqman LS-50B PCR Detection System (Perkin-Elmer, Foster City,Calif.).

An alternative method which may be used in step three to detect theamplified DNA product is ELISA detection. Depending upon the ELISAdetection method used, it may be necessary to biotinylate or otherwisemodify the primers used in step three. For example, one ELISA detectionmethod which may be used in step three is the method described byLandgraf et al. (1991, Anal. Biochem. 198: 86-91) as follows:

Primers are modified with biotinylamidocaproate-N-hydroxysuccinimidester(Sigma) and fluorescein isothiocyanate (FITC) (Sigma), by the method ofLandgraf et al. (1991, Anal. Biochem. 193: 231-235). Followingamplification the ELISA is carried out in microtiter plates coated with1 microgram/mL affinity-purified avidin (13 U/mg, Sigma). One μL of thefinal amplification product (or post-digestion product) is diluted with50 μL of PBS-Tween, and then incubated at room temperature for 30minutes in the microtiter plate well. Non-incorporated primers areremoved by washing with PBS-Tween. The plates are then incubated at roomtemperature for 30 minutes after adding 50 μL per well of anti-FITCantibody-HRPO conjugate (Dakopatts) which is at a 1:500 dilution withPBS-Tween. Following this, 80 μL of an ELISA solution made from onemilligram 3, 3′, 5, 5′ tetramethylbenzidine (Sigma) dissolved in one mLdimethyl sulfoxide, and diluted 1:10 with 50 millimol sodiumacetate:citric acid, pH 4.9, with 3 μL, of 30% (vol/vol) H₂0₂ added, isadded to each well. After 2-5 minutes, the reaction is stopped by adding80 μL of 2M H₂SO₄. The optical density is then read at 450 nm.

Alternative methods of ELISA detection which may be used in step threeinclude, but are not limited to, immunological detection methods usingmonoclonal antibody specific for RNA/DNA hybrids, such as by adaptingmethods described by Coutlee et al. (1989, Anal. Biochem. 181: 96-105),or by Bobo et al. (1990, J. Clin. Microbiol. 28: 1968-1973).

Alternative methods of ELISA detection which may be used in step threeinclude, but are not limited to, commercial detection systems such asthe SHARP signal system (Digene Diagnostics, Inc.), and the DNA enzymeimmunoassay (DEIA), (GEN-ETI-K DEIA, Sorin Biomedica).

Alternative methods by which amplified product may be detected includebut are not limited to all methods of electrochemiluminescencedetection, such as by adapting the method described by Blackburn et al.(1991, Clin. Chem. 37: 1534-1539), or by DiCesare et al. (1993,Biotechniques 15: 152-157), all methods utilizing reverse dot blotdetection technology and all methods utilizing high-performance liquidchromatography.

Finally, several separate assays examining different oncogenes ordifferent regions of the same oncogene may be performed on the samplesimultaneously, either in separate reaction tubes or, through judiciouschoice of oligonucleotides and restriction enzymes, in the same tube.This multiplexing approach allows greater sensitivity for detecting anysingle mutated oncogene and thus greater sensitivity for cancerdetection. It may be that particular patterns of mutated oncogenes, yetto be identified, have particular clinical significance as to type ofcarcinoma present or prognosis.

Therapeutic Applications

The extraction of extracellular DNA from plasma or serum, and theamplification of tumor-associated or derived DNA to detectable levels,permits further analysis or other manipulation of that DNA, from whichfurther clinical utility is realized. In this optional step of theinvention, amplified extracellular DNA is analyzed to define thecharacteristics or composition of the tumor from which the DNAoriginates. Any of several methods may be used, dependent upon thedesired information, including nucleic acid sequencing, spectroscopyincluding proton NMR spectroscopy, biochemical analysis, and immunologicanalysis. In the preferred embodiment, amplified DNA is isolated—forexample by excising mutant DNA bands from an agarose gel-reamplified,cloned into a plasmid vector, for example the pGEM-T vector plasmid(Promega) and sequenced using a commercial kit such as Sequenase 2.0(USB). Analysis to define the characteristics or composition of theextracellular DNA, and thus the tumor of origin, affords a wide array ofclinical utility, including the description, characterization, orclassification of the tumor, whether known or occult, such as by tissueof origin, by type (such as premalignant or malignant), phenotype, andgenotype, and by description or characterization of tumor behavior,physiology and biochemistry, as to gain understanding of tumorinvasiveness, propensity to metastasize, and sensitivity or resistanceto various therapies, thereby allowing the prediction of response toeither ongoing or planned therapy and, further, allowing evaluation ofprognosis. Comparison of the characteristics of extracellular DNA toprevious biopsy or surgical specimens permits further evaluation oftumor heterogeneity or similarity in comparison to that specimen, andthus evaluation of tumor recurrence.

Following extraction of extracellular DNA from plasma or serum,complimentary ribonucleic acid (RNA) may be transcribed or manufacturedfrom the DNA. In a preferred embodiment, transcription of RNA isperformed by employing a primer with an RNA polymerase promoter regionjoined to the standard primer sequence for the DNA of interest in theamplification reaction (step three). RNA complimentary to the DNA isthen transcribed from the attached promoter region. In an alternativemethod, amplified extracellular DNA is cloned into an expression vector,and RNA complimentary to the DNA is transcribed. Furthermore, as anoptional preferred embodiment, the complimentary RNA is used in an invitro translation reaction to manufacture tumor-associated ortumor-specific protein.

Extraction of extracellular DNA, amplification of tumor-derived ortumor-associated DNA, and characterization, transcription ofcomplimentary RNA, and translation to tumor-associated or tumor-specificprotein, provides significant utility, both in the assignment of therapyand in the development of tumor-specific therapies. Sequencing ofextracellular DNA or transcription of complementary RNA allowsassignment or development of antisense compounds, including syntheticoligonucleotides and other antisense constructs appropriately specificto the extracellular DNA, such as by construction of an expressionplasmid such as by adapting the method of Aoki et al. (1995, Cancer Res.55: 3810-3816). Similarly, defining tumor characteristics allowsassignment of specific monoclonal antibody or vaccine therapiesappropriately specific to the amplified DNA. Production of correspondingimmunologic protein can be used in the development of tumor-specificmonoclonal antibodies. Similarly, translated protein can be used intumor-specific vaccine development. Furthermore, the extracellular DNApermits a means of defining or allowing the construction of a DNAconstruct which may be used in vaccine therapy.

Of particular value, the invention allows the development andapplication of these tumor-specific therapies even when onlypremalignant tumors, early cancers, or occult cancers are present. Thus,the invention allows therapeutic intervention when tumor burden is low,immunologic function is relatively intact, and the patient is notcompromised, all increasing the potential for cure.

The invention also provides methods for transcribing RNA complementaryto the isolated extracellular nucleic acid from plasma or serum, as wellas methods for producing peptides and proteins (or fragments thereof)encoded thereby. Additional methods for using the peptide and proteinsas antigens for producing antibodies specific for the peptides andproteins encoded by the extracellular nucleic acids of the invention arealso provided. The isolated extracellular nucleic acids of the inventionare also used in methods for producing antisense oligonucleotides,either synthetically or using recombinant genetic methods, and the usethereof for affecting gene expression in a cell will be appreciated byone having ordinary skill in the art in view of the methods forisolating and identifying said extracellular nucleic acid providedherein. Vaccine production, as is understood by one with skill in theart, is also enabled using the methods of the invention.

The methods of the invention and preferred uses for the methods of theinvention are more fully illustrated in the following Examples. TheseExamples illustrate certain aspects of the above-described method andadvantageous results. These Examples are shown by way of illustrationand not by way of limitation.

EXAMPLE 1

Detection of Extracellular Mutant K-ras Oncogene DNA in Plasma or Serum

1. Background

Colorectal cancer (CRC) is a common and often fatal disease,representing the second or third leading cause of cancer death in theU.S. Local spread of disease is common, and regional or widespreadmetastasis has occurred in roughly 60% of CRC at the time of diagnosis(Parker et al., ibid.). Current screening tests for CRC involve stoolsampling for occult blood or endoscopic examination. These methodsprovide no information on the spread of disease, however.

Advances in the understanding of the benign-to-malignant transformationsequence are based largely on studies of CRC and its precursors (Fearonet al., 1987, Science 238: 193-197; Fearon & Vogelstein, 1990, Cell 61:759-767; Hamilton, 1992, Cancer 70: 1216-1221). The genesis of anadenocarcinoma is understood to require the occurrence of a number ofmutational events, leading to the transformation of normal epitheliuminto a clonal malignancy. While no single event has been identified asbeing crucial to the development of CRC, mutation of the K-ras oncogenehas been detected in 40-75% of all CRC and is found in roughly the sameproportion of pre-malignant adenomas (Bos et al., 1987, Nature 327:293-7; Yamagata et al., 1994, Jap. J Cancer Res. 85: 147-51). K-rasmutation occurs in later stages of adenoma development and persistsduring the clonal transformation process (Vogelstein et al., 1988, N.Engl. J. Med. 319: 525-32). Mutations of K-ras, located on chromosome12p, seem to play an important role in a number of malignancies. In CRCthese mutations almost always are missense mutations confined to codons12, 13 and 61, with the first in particular being common (Chaubert etal., 1994, Amer. J. Path. 144: 767-75; Kondo et al., 1993, Cancer 73:1589-94; Oudejans et al., 1991, Int. J. Cancer 49: 875-9; Pellegata etal., 1992, Anticancer Res. 12: 1731-6; Sidransky et al., 1992, Science256: 102-5). These mutations appear to alter the normal function of thisproto-oncogene (Finney & Bishop, 1993, Science 260: 1524-7; Shirasawa etal., 1993, Science 260: 85-8).

2. Details of the Assay

This embodiment of the inventive assay was performed in the followingsteps.

Step one: Plasma or serum samples from six patients with advancedcolorectal cancer and 15 normal volunteers were used in the assay.Extracellular DNA from plasma or serum samples was co-precipitated withgelatin using a modification of the method of Fournie et al. (1986,Anal. Biochem. 158: 250-6). Briefly, 160 μL plasma or serum was mixedwith 12.8 μL 0.5 M EDTA and 467 μL sterile, double-distilled water, thenemulsified for 3 minutes with 320 μL phenol or phenol:chloroform:isoamylalcohol (25:24:1). The solution was centrifuged at 14,000×g for 10minutes to resolve the aqueous and organic phases, and 570 μL of theaqueous layer was removed to a clean tube. DNA was precipitated byaddition of 142 μL of a 0.3% gelatin solution prepared as describedabove and 500 μl, of cold absolute ethanol, followed by incubation at−20° C. for 2 hours. The sample was centrifuged at 14,000×g at 6° C. for15 minutes, washed once with cold 70% ethanol, and dried in a 60° C.heat block for 10 minutes. DNA was then recovered by the addition of 35to 70 μL of sterile, double-distilled water preheated to 60° C.Thirty-five μL of the resuspended DNA was used in the second step of theassay.Step two: DNA fragments specific for nucleic acid sequences of mutantK-ras oncogene in the isolated extracellular DNA preparation of Step Onewere amplified utilizing a non-radioactive PCR assay adapted from Kahnet al. (1991, Oncogene 6: 1079-1083) as follows. A reaction mixture wasprepared containing 35 μL of the isolated extracellular DNA of Step One,50 mM potassium chloride, 10 mM Tris buffer (pH 9.0), 0.1% Triton X-100,1.5 mM magnesium chloride, 200 μM for each nucleoside triphosphate(dATP, dGTP, dCTP, and dTTP), 0.5 pmol oligonucleotide K-ras primer 1having the sequence:

5′-ACTGAATATAAACTTGTGGTAGTTGGACCT-3′, (SEQ ID No.1)0.75 pmol oligonucleotide K-ras primer 2 having the sequence:

5′-TCAAAGAATGGTCCTGGACC-3′, (SEQ ID No.:2)and 1 U Taq DNA polymerase (Promega, Madison, Wis.) in a final volume of50 μL. Oligonucleotide K-ras primer 1 was constructed to contain thenucleotide sequence that is immediately upstream of mutant codon 12(positions 99-128; Genbank Accession #L00045) of the K-ras gene, and ismodified at the 28th base (G-C) to create a non-naturally-occurringrestriction enzyme digestion site (BstNI). Oligonucleotide K-ras primer2 is constructed to contain the nucleotide sequence complementary to thesequence of K-ras (at the complement of positions 255-236; GenbankAccession #L00045), and is modified at the 17th nucleotide (C→G) tocreate a non-naturally-occurring BstNI site that serves as an internalcontrol to monitor restriction enzyme digestion. The reaction mixturewas overlaid with mineral oil and thermocycled 15-20 times using athermal profile of 94° C. for 48 seconds, 56° C. for 90 seconds, and 72°C. for 155 seconds in a PHC-2 thermocycler (Techne, Princeton, N.J.).Ten μL of the PCR mixture was then removed to a new tube and mixed with1× BstNI reaction buffer and 10 units BstNI restriction enzyme(Stratagene, La Jolla, Calif.), and then incubated at 60° C. for 90minutes. A second aliquot of 10 units BstNI was added and the reactioncontinued for an additional 90 minutes.Step three: Ten μL of the digested PCR mixture was removed to a cleantube and a new reaction mixture was set up for the second round ofamplification, using the same constituents as in the first amplificationwith the exception that 7.7 pmoles of oligonucleotide K-ras primer 1 and11.5 pmoles of oligonucleotide K-ras primer 2 were used. The samecycling conditions were employed for 33-35 amplification cycles. Asecond BstNI restriction digestion was then performed using 25 μL of thesecond step PCR product and 17 units of enzyme in a final volume of 35μL. Digestions were performed for 60 min at 60° C., followed by theaddition of a second aliquot of 10 U of enzyme and a digestion for anadditional 60 min. The final digestion product was analyzed by gelelectrophoresis on a 3% agarose gel (NuSieve, FMC Bioproducts, Rockland,Me.) in 1×TBE buffer at 75V DC for about 2 h and DNA fragmentsvisualized by staining with ethidium bromide and ultraviolet lightillumination (Foto-prep Transilluminator, Fotodyne, Hartland, Wis.).

All amplification assays included as a positive control DNA from a coloncarcinoma cell line GEO known to contain a GGT→GCT mutation in codon 12of the K-ras gene; a negative control containing wildtype K-rassequences consisting of normal placenta tissue, and a negative controlfor PCR contamination consisting of water substituted for DNA in thereaction mixture. In addition, reactions were run in parallel withoutBstNI digestion to ensure amplification had occurred (as shown in FIG.2). Routine precautions to prevent PCR contamination were employed inall amplification-based assays. The risk of contamination yieldingfalsely positive results was further minimized by repeating PCR assayson all patient plasma or serum samples 2-3 times on different days.

Following gel electrophoresis, DNA fragments of the expected size wereexcised, reamplified, cloned into the pGEM-T vector (Promega), and thenucleotide sequence determined using a commercial sequencing kit(Sequenase 2.0, USB, Cleveland, Ohio). A minimum of two clones weresequenced for each PCR. Of the 6 patients with colorectal cancer, K-rasmutations were detected in the plasma or serum of 4 (67%) (shown in FIG.3). The blood of all normal volunteers tested negative for K-rasmutations (shown in FIG. 4). In prototype experiments and using patientplasma or serum samples, this assay has been shown repeatedly to have asensitivity capable of detecting 1 mutant K-ras molecule equivalent in abackground of 100,000 to 1,000,000 wildtype K-ras molecules. However, tomake certain that negative results were not due to failedamplifications, specimens were further tested by omitting the initialBstNI step digestion. In these experiments, a DNA fragment correspondingin size to the expected wildtype K-ras fragment was found in all cases(data not shown). In addition, the positive GEO control tested positive,and the negative placenta and water blank controls tested negative, inall PCR assays.

The above example describes detection of mutant K-ras in plasma or serumfrom patients with colorectal cancer, and the same methods are employedto detect K-ras mutations in plasma or serum from patients having anycancer associated with K-ras mutations, including colorectal, lung,pancreatic, and gastric cancers.

3. Premalignant Disease Detection Using Card

In addition, the methods described herein can be used to detectpremalignant or occult solid tumor disease. For example, a familyhistory of colorectal cancer is a significant risk factor for thedevelopment of colorectal cancer, particularly if family historyincludes early onset.

Use of the CARD assay of the invention to detect extracellularoncogene-related DNA was performed on such a patient as follows. Plasmawas collected from a 28 year old woman with recent rectal bleeding and afamily history of colorectal cancer (one aunt who had died in earlyadulthood from colorectal cancer). The patient had undergone endoscopyand colonoscopy at the time of plasma collection, and had no clinicalevidence of colorectal cancer. The patient's plasma was thereforesubjected to CARD analysis for the detection of extracellular DNArelated to the K-ras oncogene.

This assay was performed as follows:

Step one: Plasma DNA was co-precipitated with gelatin and recovered asdescribed above.Combined Steps two and three: A reaction mixture was prepared containing35 μL of the extracted DNA solution, 50 mM KCl, 10 mM Tris-HCl (pH 9),0.1% Triton X-100, 1.5 mM MgCl₂, 200 μM each dNTP, 15 pmol each of K-rasprimers 1 and 2 (SEQ ID Nos.: 1 & 2), 4 U BstNI restriction endonuclease(Stratagene, LaJolla, Calif.) and 1 U Taq polymerase (Promega, Madison,Wis.). This reaction mixture was overlaid with mineral oil andthermocycled using a protocol wherein the reaction was incubated at 94°C. for 7 seconds, then at 60° C. for 3 min, then at 94° C. for 5seconds, then at 60° C. for 3 min, and so on, so that at each cycle thedenaturation time at 94° C. decreased by one second until the seventhcycle (having a denaturation time of 1 second), which was repeated foran additional 33 cycles, for a total of 40 cycles in the amplificationreaction. After cycle 10, thermocycling was paused at 60° C. and anadditional 10 U of restriction enzyme added.

At the completion of the thermocycling reaction, 20 μL of theamplification/digestion mixture were removed into a fresh reaction tubeand mixed with 10 U BstNI in the appropriate buffer to a total volume of30 μL and incubated at 60° C. for 60 min. A second 10 U aliquot of BstNIwas added and the reaction incubated for an additional 60 min at 60° C.

This digestion reaction product was analyzed by gel electrophoresis on a3% agarose gel; the results of this analysis are shown in FIG. 6. Allassays included a K-ras positive control comprising of DNA from a coloncarcinoma cell line containing K-ras having a codon 12 mutation; a K-rasnegative control consisting of normal placental tissue DNA; and anegative control for PCR contamination, comprising a water blank.Reactions were also performed in parallel without BstNI digestion as acontrol for PCR amplification. Routine precautions associated withamplification were employed in all amplification-based experiments.

The results of this assay, as shown in FIG. 6 demonstrated the presenceof mutant K-ras extracellular DNA in the patient plasma sample (lane 7).These results demonstrate that the methods of the invention enables thedetection of extracellular DNA related to mutated oncogenes associatedwith occult or premalignant solid tumor disease. The detection ofextracellular DNA encoding a mutant K-ras oncogene known to beassociated with colorectal cancer in a patient having no clinical signsor symptoms of colorectal neoplasia demonstrates that the instant assayincreases diagnostic assay sensitivity and ability to detectpremalignant or occult neoplasia early in the course of the disease. Theability of the methods of the invention to detect tumor-related DNA frompremalignant or occult neoplastic disease patients provides the capacityto better direct prevention, early detection, intervention, monitoringand management of neoplasia and pre-neoplastic disease, and affords theopportunity for medical intervention earlier in the disease course thanheretofore, increasing the likelihood of success of treatment and cure.

EXAMPLE 2 Detection of Extracellular Bcl-2 DNA and Bcl-2/IgHTranslocations in Plasma or Serum 1. Background

Follicular center cell lymphoma (follicular lymphoma) is the most commonform of primary malignancy of the lymph nodes in the U.S., comprisingmore than half of all cases of lymphoma. Follicular lymphoma isgenerally a slowly progressive malignancy, with patient survivalaveraging several years to a decade or more. Standard treatment offollicular lymphoma depends on factors such as extent of disease andage, and typically involves multi-agent chemotherapy. Newer approachesto therapy include high dose chemotherapy with bone marrowtransplantation and immunotherapy, either actively or passively induced.Because of the high rate of relapse among patients treated with standardregimens, and because of the general oncologic tenet that treating smallamounts of tumor rather than large masses is more efficacious, there isa need for methods to detect minimal amounts of tumor in follicularlymphoma patients.

Follicular lymphomas are distinguished by a particular geneticalteration, the breaking and rejoining of chromosomes 14 and 18 to eachother. This breaking, or translocation, results in the juxtaposition oftwo genes in a head-to-tail fashion: the oncogene bcl-2 on chromosome18, which is known to play a role in the control of programmed celldeath; and an immunoglobulin heavy chain gene (IgH) on chromosome 14.Uniting these two genes as a result of translocation causes adysregulation of the bcl-2 gene. This is thought to be due to the factthat immunoglobulin heavy chain genes are typically activated in thelymphoid cells from which this malignancy derives, and in thetranslocation the adjacent bcl-2 gene inappropriately shares in theactivation. Translocation occurs in approximately 80-90% of follicularlymphomas, and in two-thirds to three-quarters of these cases thetranslocation involves one of two well-characterized sites. The sites offrequent bcl-2 breakage fall within small areas (a few hundred basepairs) termed the Major Breakpoint-cluster Region (MBR) and the minorbreakpoint-cluster region (mcr). The translocation at the immunoglobulinlocus also occurs in restricted regions, since the breakage mimics thenormal immunoglobulin gene rearrangement process. The restricted natureof the translocation permits prediction in most cases of the DNAflanking the breakpoints, which thereby provides diagnostic nucleic acidfragments uniquely found in cells having this translocation.

2. Details of the Assay

The assay was performed in the following steps.

Step one: Four patients with follicular lymphoma had serum drawn priorto or early in a standard course of antineoplastic chemotherapy. Threeof these patients had been previously demonstrated by PCR to have tumorcells containing a MBR translocation; the fourth patient had noPCR-detectable MBR or mcr translocation in tumor cells. Serum DNA wasco-precipitated with gelatin and recovered as described above in Example1.Step two: Tumor specific bcl-2/IgH translocations were amplified using anon-radioactive PCR assay as follows. A reaction mixture was preparedcontaining 35 μL, of the extracted DNA solution of Step One, 50 mMpotassium chloride, 10 mM Tris buffer (pH 9.0), 0.1% Triton X-100, 1.5mM magnesium chloride, 200 μM of each nucleotide triphosphate (dATP,dGTP, dCTP, and dTTP), 1 pmol oligonucleotide MBR comprising thesequence:

5′-TTAGAGAGTTGCTTTACGTG-3′, (SEQ ID No.:3)1 pmol oligonucleotide J_(H)(CON) comprising the sequence:

5′-ACCTGAGGAGACGGTGACC-3′, (SEQ ID No.:4)in a total volume of 48 μL. One Unit of Taq DNA polymerase (FisherChemical Co., Fairlawn, N.J.) diluted to 2 μL in the same buffer wasadded to each sample after samples had been pre-heated to 95° C.

The oligonucleotide MBR was constructed to contain the nucleotidesequence that is immediately upstream of the most frequent site oftranslocation in the bcl-2 gene (positions 4415-4434; Genbank Accession#I08038). The oligonucleotide J_(H)(CON) is constructed to containconsensus sequences to the 3′ ends of the 6 J_(H) segments of theimmunoglobulin heavy chain gene, and will hybridize with each J_(H)segment under the conditions of PCR amplification used herein. Thus,translocation of the bcl-2 gene into any of the J_(H) regions permitsspecific and exponential amplification from the involved region using anupstream translocation primer such as MBR.

The reaction mixture was cycled 20 times using a thermocycling profileof 94° C. for 1 minute, 56.5° C. for 2 minutes, and 72° C. for 3 minutesin a Deltacycler thermocycler (Ericomp, San Diego, Calif.).

Step three: Two μL of the PCR mixture of Step Two was removed to a cleantube and a new reaction mixture was set up for the second round ofamplification using the same components of the amplification reactionmixture as described above in Step Two, with the exception that 25pmoles of oligonucleotide MBR-int having the sequence:

5′-GCCTGTTTCAACACAGACC-3′ (SEQ ID No.:5)(positions 4435-4453; Genbank Accession #I08038) and 25 pmoles ofoligonucleotide J_(H)(CON) (SEQ ID No.: 4) were used. The MBR-int primerlies internal to the MBR primer and increases the specificity of thesecond round of amplification. The same cycling conditions as above wereemployed for a total of 30 amplification cycles. The final amplificationreaction products were analyzed by gel electrophoresis on a 3% agarosegel (NuSieve) in TBE buffer.

All amplification assays included a bcl-2/IgH positive controlconsisting of the lymphoma cell line MB-1 containing a diagnostic andwell-characterized breakpoint (comprising the sequence at position 3110of bcl-2: GTT . . . ctc . . . GGATTGGACG translocated into the J_(H) (6)immunoglobulin heavy chain gene, where N-nucleotide additions orN-insertions are in lower case and somatic mutations are underlined); awildtype bcl-2/IgH negative control consisting of normal placentatissue, and a PCR contamination negative control consisting of watersubstituted for DNA. Routine precautions to prevent PCR contaminationwere employed in all amplification-based work.

Results from these assays are shown in FIG. 5. For the three patientswith bcl-2/IgH translocations detectable in their tumor cells, identicalDNA fragments corresponding to hemi-nested PCR amplification weredetected in the extracellular DNA isolated from serum. The patientwithout a detectable translocation in tumor cell DNA (due to either lackof a translocation or a variant translocation not detectable with theseprimers) did not have a translocation-specific PCR product detectable inserum. All controls produced the expected fragments (or lack offragments for the PCR negative controls).

A similar assay is used to detect bcl-2 sequences per se (i.e., withoutassaying for a specific translocation breakpoint within the bcl-2 gene).This is accomplished using a 3′ PCR primer constructed to comprise thecomplement of a nucleotide sequence of bcl-2 at a defined distance 3′ tothe bcl-2 specific primer described above (SEQ ID No.: 3), substitutedfor the J_(H)(CON) primer described above (SEQ ID No.: 4) in PCRamplification reactions performed as described herein. This bcl-2specific primer has the sequence

5′-GGAGGATCTTACCACGTGGA-3′. (SEQ ID No.:10)

PCR amplification using this pair of bcl-2 specific amplificationprimers are useful for detecting extracellular DNA in patient serum orplasma independent of the specific translocation associated withlymphoma, and thus provides a method for detecting putativelymphoma-bearing patients not bearing well-characterizedlymphoma-specific translocations, and for detecting bcl-2 relatedextracellular DNA associated with other (non-lymphoma) cancers.

EXAMPLE 3 Detection of Extracellular Mutant P53 DNA in Plasma orSerum 1. Background

The p53 oncogene is one of the most frequently mutated tumor suppressorgenes in human cancer. Among other functions, it is a regulator of thecell cycle and is involved in programmed cell death, and its mutationpermits unopposed cell proliferation. In colorectal cancer (describedabove in Example 1), approximately half of all CRCs contain mutations ofthe p53 oncogene (Greenblatt et al., 1994, Cancer Res. 54: 4855-78), anda survey of the EMBL Data Library of 360 published mutations of p53 inCRC cases indicates that 49% occur at 5 particular amino acids(Hollstein et al., 1994, Nucleic Acids Res. 22: 3551-5). Recent dataindicate that p53 and K-ras mutations in CRC tend to be mutuallyexclusive, that is, tumors are commonly found with only one or theother, rarely both (Dix et al., 1995, Diagn. Molec. Path. 4: 261-265).These finding suggested that an assay for p53 gene mutations inextracellular DNA in serum or plasma would identify patients other thanthose identified using the K-ras assay described above in Example 1. Thep53 mutational “hot spots” in colorectal cancer are amino acids 175,245, 248, 273, and 282. This clustering of mutations may permit a panelor multiplex approach to the amplification-based assays disclosed hereinusing a number of primer pairs and restriction enzymes to identifyaffected patients (illustrated in Tables I-III below). Although CRC isexemplified in this Example, one of ordinary skill will appreciate thatany other malignancy having p53 mutations can be analyzed using theassays of the invention.

2. Details of the Assay

The assay was performed using the following steps.

Step one: Extracellular DNA from patient plasma or serum isco-precipitated with gelatin as described above in Example 1.Step two: Nucleic acid comprising mutant p53 oncogene sequences areamplified utilizing a non-radioactive PCR assay performed as follows. Areaction mixture is prepared as described above, containing 35 μL of theextracted extracellular DNA from plasma or serum, 50 mM potassiumchloride, 10 mM Tris buffer (pH 9.0), 0.1% Triton X-100, 1.5 mMmagnesium chloride, 200 μM apiece of each deoxynucleoside triphosphate(dATP, dGTP, dCTP, and dTTP), and the following pairs and amounts of theprimers shown in Table I:0.7 pmol exon 5 oligonucleotide 5′-primer

5′-GCAGTCACAGCACATGACG-3′ (SEQ ID No.:11)and0.5 pmol exon 5 oligonucleotide 3′-primer

5′-AATCAGAGGCCTGGGGAC-3′; (SEQ ID No.:12)or0.7 pmol exon 7 oligonucleotide 5′-primer

5′-GGGCCTGTGTTATTCTCCTAGG-3′ (SEQ ID No.:13)and0.5 pmol exon 7 oligonucleotide 3′-primer

5′-CCAGTGTGATGATGGTGAGG-3′; (SEQ ID No.:14)or0.5 pmol exon 8 oligonucleotide 5′-primer

5′-GGACGGAACAGCTTTGAGGCG-3′ (SEQ ID No.:15)and0.5 pmol exon 8 oligonucleotide 3′-primer

TCCCCGGGGGCAGCGCGT; (SEQ D No.:16)and 1 Unit of Taq DNA polymerase (Fisher) in a final volume of 50 μL.One member of each primer pair is prepared having a sequencemodification to create a non-naturally-occurring restriction enzyme sitefor the corresponding enzyme in Table I to serve as an internal control.Each of the activating mutations for each primer pair shown in Table Idestroy a restriction enzyme site normally found in the p53 gene,thereby permitting enrichment of the samples for the mutant allele byrestriction enzyme digestion prior to second round amplification (asdescribed in Example 1). The amplification reaction mixture isthermocycled 15-20 times using a protocol of 94° C. for 48 seconds, 57°C. for 90 seconds (for exon 5 or 7 primers) or 61° C. for 90 seconds(for exon 8 primers), and 72° C. for 155 seconds in a Deltacyclerthermocycler (Ericomp).

After amplification, 10 μL of the PCR mixture is removed to a clean tubeand mixed with 1× reaction buffer and 10 units of the appropriaterestriction enzyme for each primer pair shown in Table I, and incubatedat the appropriate temperature for 90 min. A second aliquot of 10 unitsof restriction enzyme is added and the reaction continued for anadditional 90 min.

Step three: Ten μL of the digested PCR mixture is transferred to a cleantube and a new amplification reaction mixture is prepared for a secondround of amplification using the same constituents as in the firstamplification, except that 35 pmoles of oligonucleotide 5′-primer (exons5 and 7) or 25 pmoles of oligonucleotide 5′-primer (exons 8) and 25pmoles of oligonucleotide 3′-primer (exons 5, 7 and 8) are used. Thesame thermocycling conditions are employed for 33-35 amplificationcycles. A second restriction digestion is performed using 25 μL of thesecond step PCR product and 17 units of enzyme in a final volume of 35μL. Digestions are performed for 60 min, followed by the addition of asecond aliquot of 10 units of enzyme and a final digestion for anadditional 60 min. The final digestion product was analyzed by gelelectrophoresis on a 3% agarose gel (NuSieve) in TBE buffer. In thepractice of this invention the production of elevated levels ofnon-specific DNA fragments produced by PCR may be anticipated.Hybridization with detectably-labeled specific probes may therefore beused to increase assay sensitivity.

The expected sizes of the wildtype, undigested PCR product DNA fragmentsare 130 bp (exon 5), 111 bp (exon 7) and 104 bp (exon 8). DNA fragmentscorresponding to wildtype alleles for exon 5 are cleaved to 79, 33, and18 bp by HhaI, and mutation at p53 position 13103 changes the digestedfragment sizes to 79 and 51 bp. DNA fragments corresponding to wildtypealleles for exon 7 are cleaved to 7, 85, and 19 bp by MspI, and mutationat p53 positions 14069 or 14070 yields fragment sizes 7 and 104 bp. DNAfragments corresponding to wildtype alleles for exon 7 are cleaved to82, 22, and 7 bp by AciI, and mutation at p53 positions 14060 or 14061yields fragment sizes 104 and 7 bp. DNA fragments corresponding towildtype alleles for exon 8 are cleaved to 19, 76, and 9 bp by BstNI,and mutation at p53 positions 14486 or 14487 yields fragment sizes 95and 9 bp. DNA fragments corresponding to wildtype alleles for exon 8 arecleaved to 47, 52, and 5 bp by MspI, and mutation at p53 positions 14513or 14514 yields fragment sizes 99 and 5 bp. The combination of specificamplification using the p53 primers described in Table I, digestion withthe appropriate restriction enzymes and detection of DNA fragments ofthe expected sizes results in detection of extracellular DNA in plasmaor serum corresponding to any of the expected mutant alleles of p53.

Alternatively, in a preferred embodiment, amplification can be performedusing hemi-nested primers as shown in Table II that can provide morespecific and rapid results in Step Three of the methods of theinvention. In this assay, extracellular DNA is extracted as describedabove from patient plasma or serum. PCR amplification reactions areperformed as described above, with the exception that 1 pmol of each ofthe appropriate external primers are used in the first PCR amplificationreaction, these primers being described in Table II. The pattern of PCRprimer utilization in these assays is summarized in Table m, wherein thefirst amplification is performed with the primer pairs labeled “StepTwo” and the second amplification is performed with the primer pairslabeled “Step Three.” For the first amplification reaction, thethermocycling protocols used is a total of 15 amplification cycles of94° C. for one minute, 59° C. for two minutes, and 72° C. for twominutes in a thermocycler. All primer combinations described use thissame cycling protocol. Upon completion of the first amplificationreaction, 10 μL of the PCR mixture is removed to a clean tube and mixedwith 1× reaction buffer and 10 Units of the appropriate restrictionenzyme shown in Table I, and incubated at the appropriate temperaturefor 60 min. A second aliquot of 10 Units of restriction enzyme is addedto each reaction and digestion continued for an additional 60 min.

The third step in the assay is an additional amplification reactionusing the Step Three amplification primer pairs as shown in Table III.For these reactions, 10 μL of the digested PCR mixture is removed to aclean tube and a new amplification reaction mixture constructed asdescribed above, substituting 25 pmoles of each of the Step Three primerpairs in Table III for the Step Two primer pairs used in the firstamplification reaction. Thermocycling is performed for 33-35amplification cycles under the same conditions as described above, withthe exception that the annealing temperature for exon 5 and exon 7primers is 58° C. and the annealing temperature for exon 8 primers is60° C. After completion of the amplification reaction, a secondrestriction digestion is performed using 25±μL of the Step Three PCRproduct and 17 units of enzyme in a final volume of 35 μL. DNA fragmentsare digested for 60 min, followed by the addition of a second aliquot of10 Units of enzyme and a final digestion for an additional 60 min. Thefinal digestion product was analyzed by gel electrophoresis on a 3%agarose gel (NuSieve) in TBE buffer. The expected sizes of the DNAfragments obtained for the wildtype and mutant p53 alleles assayed usingthis method are those described above.

Detection of extracellular DNA in patient plasma or serum may beachieved using sera or plasma from patients having any cancer associatedwith p53 mutations, including cancers of the colon, rectum, bladder,breast, esophagus, liver, lung, cervix, and brain, and sarcomas,lymphomas, leukemias, and melanomas. In particular, mutations in aminoacid 249 are found more frequently in liver cancer and lung cancer thanin any other primary site. This mutation also forms a large

TABLE I p53 mutation “hotspots” in CRC and assay reagents for detectingmutations Activating mutation(s) amino acid exon 5′ primer sequence3′ primer sequence Enzyme 13103 175 5 GCAGTCACAGCACATGACGAATCAGAGGCCTGGGGAC HhaI 14060, 14061 245 7 GGGCCTGTGTTATTCTCCTAGGCCAGTGTGATGATGGTGAGG Aci I 14069, 14070 248 7 GGGCCTGTGTTATTCTCCTAGGCCAGTGTGATGATGGTGAGG MspI 14486, 14487 273 8 GGACGGAACAGCTTTGAGGCGTCCCCGGGGGCAGCGCGT BstUI 14513, 14514 282 8 GGACGGAACAGCTTTGAGGCGTCCCCGGGGGCAGCGCGT MspI

TABLE II p523 hemi-nested primers for amplifying mutation “hotspots” inCRC Mutation(s) External primer 5′ primer sequence 3′ primer sequence13103 GGGCCAGACCTAAGAGCAAT GCAGTCACAGCACATGACG AATCAGAGGCCTGGGGAC 14060,14061 GCCTCCCCTGCTTGCCAC GGGCCTGTGTTATTCTCCTAGG CCAGTGTGATGATGGTGAGG14069, 14070 GCCTCCCCTGCTTGCCAC GGGCCTGTGTTATTCTCCTAGGCCAGTGTGATGATGGTGAGG 14486, 14487 CTGATTTCCTTACTGCCTCTTGCTTGGACGGAACAGCTTTGAGGCG TCCCCGGGGGCAGCGCGT 14513, 14514CTGATTTCCTTACTGCCTCTTGCTT GGACGGAACAGCTTTGAGGCG TCCCCGGGGGCAGCGCGT *Mutation numbering after Genbank Sequence X02469, Hsp53

TABLE III Combinations of Nested Primer Pairs for p53 AmplificationActivating Primers used in Step Primers used in Step mutation(s) TwoThree 13103 External + 5′ 5′ + 3′ 14060, 14061 External + 3′ 5′ + 3′14069, 14070 External + 3′ 5′ + 3′ 14486, 14487 External + 3′ 5′ + 3′14513, 14514 External + 3′ 5′ + 3′proportion of all p53 mutations in those tumors (28% in liver, 6% inlung) (Hollstein et al., 1994, ibid.). This may be due to particularcarcinogenic susceptibility of these organs. This fact may permit theuse of a relatively specific assay to detect particular primary cancersin patients at risk, e.g., patients with cirrhosis who have an elevatedrisk of liver cancer, or smokers predisposed to lung cancer.

EXAMPLE 4 Prophetic Examples of the Use of the Assays of the Invention

The following examples are illustrative of clinical uses for the assaysof the invention.

Case 1

A 26 year old asymptomatic man presents for evaluation after learninghis 37 year old brother was recently diagnosed with colon cancer.Peripheral blood is drawn in order to evaluate the patients plasma forthe presence of extracellular mutant K-ras DNA. Plasma extracellular DNAis extracted by the gelatin extraction method as described, followed byPCR amplification using K-ras primers with diagnostic restriction enzymedigestion sites as described. To increase the sensitivity of the assay,a two-step amplification assay is performed with digestion of PCRproducts in Step Two, followed by reamplification and final digestion inStep Three. The final digestion product is analyzed by gelelectrophoresis on a 3% agarose gel for detection of mutant-specific DNAfragments. The presence of these DNA fragments in the patient's plasmaindicates that mutant K-ras extracellular DNA is present in thepatient's blood plasma. K-ras oncogene mutations are present in 40-50%of colon cancer, initially occurring in the premalignant adenoma stage,but persisting throughout transformation to frank malignancy andmetastatic colon cancer. Although colon cancer is highly curable ifdiagnosed at an early stage, it is fatal when diagnosed at advancedmetastatic stages. The positive results of the assay of the inventionfor this patient, in a setting of a strongly positive family history forcolon cancer, are highly suggestive of either premalignant or malignantcolon cancer. Such a patient would be advised to undergo colonoscopy,and if no lesion is found, to receive surveillance more frequently thanwould normally be given.

This hypothetical case illustrates how the invention can be used as alow-cost means for identifying patients at high-risk for cancer,specifically colon cancer, and to discriminate between such patients whoshould receive further, more aggressive and more expensive preventivecare from those at lower risk who do not require such additionalsurveillance. The assay of the invention provides for the detection ofeither premalignant or malignant conditions prior to the metastaticstate, and can thus play a role in clinical management of human cancer.Because K-ras mutations are also noted in other cancers, such aspancreatic and lung cancer, additional amplification reactions using amultiplex panel approach to detect multiple different tumor-associatedextracellular DNAs, including for example p53, DCC, and APC DNA, permitsa more exact discrimination of the potential tissue source ofextracellular mutant oncogene DNA in plasma and serum, and informsclinical efforts for farther diagnostic interventions includingdirecting such efforts to those tissues most likely to comprise anoccult neoplasm, while at the same time having the potential toeliminate the need for unnecessary screening of a variety of othertissues for neoplasia based on a failure to detect the appropriatecollection of related extracellular DNAs.

Case 2

A 33 year old woman sees her local dermatologist after noting a“bleeding mole” on her back. Local excision diagnoses a malignantmelanoma of 0.3 millimeter depth. Wide surgical re-excision isperformed, and the patient is told she is likely cured and no furthertherapy is needed. Molecular analysis of the resected melanomademonstrates that it is a mutant p53-positive tumor. Three monthsfollowing her surgery the patient seeks a second opinion regarding theneed for further therapy. Peripheral blood is drawn to evaluate thepatient's plasma for the presence of extracellular mutant p53 oncogeneDNA using the assays of the invention. Extracellular DNA is extractedfrom plasma as described above using the silica extraction method,followed by PCR amplification for extracellular mutant p53 DNA.p53-specific amplification products are detected by ELISA. In this case,the inventive assay detects the presence of mutant p53 in the patient'splasma matching the mutation found in the original tumor, with thepresence of this DNA in plasma indicating latent malignant melanoma.Consequently, the patient is started on adjuvant therapy withinterferon-alpha. Extracellular plasma p53 oncogene DNA levels aresubsequently followed in a quantitative fashion using the assays methodsof the invention. Blood is drawn from the patient every two months, andextracellular plasma DNA is extracted and analyzed by quantitative PCRamplification for mutant p53 DNA using biotinylated primers and anelectrochemiluminescence-based detection means. Invention datademonstrate a rise in the patient's mutant p53-specific extracellularplasma DNA levels. As a consequence, interferon treatment is stopped,and the patient is enrolled into an experimental adjuvant therapyprotocol.

This hypothetical case illustrates several uses of the invention,including the detection of latent cancer, prediction of diseaseprognosis and cancer recurrence following surgical excision,determination of the need for additional therapy, evaluation of thebenefit of therapy and the need to change therapies, and furtherevaluation of the prognosis of patients as a result of therapy.

Case 3

A 76 year old man is found to have a pancreatic mass on CT scan imaging.H is chest x-ray and colonoscopy are normal. The patient refuses toconsider surgery because of the significant surgical risks. He elects toreceive patient-specific therapy made possible by use of the invention.Since K-ras mutations are present in 80-90% of pancreatic cancers,peripheral blood is drawn to evaluate the plasma and characterizeextracellular mutant K-ras DNA circulating in plasma using the assaymethods of the invention. Extracellular DNA in plasma is extracted usingthe gelatin method as described, followed by PCR amplification andanalysis of PCR products by agarose gel electrophoresis. Mutant K-rasamplification products are excised from the gel and the sequence of theK-ras specific fragment determined using a commercial kit. Detection ofmutant K-ras sequences support the likelihood of the pancreatic massbeing malignant. On the basis of the mutation sequence, apatient-specific therapy (i.e., specific to the patient's own cancer) isdeveloped, in this case a ras vaccine specific to the mutant oncogene inthis patient's pancreatic cancer.

In this hypothetical case, the invention is used not only to helpconfirm a suspected diagnosis of pancreatic cancer, but to develop apatient-specific therapy. Patient-specific therapies—i.e., therapiesspecifically designed for a given patient's cancer, or a given type ofcancer—are possible when specific characteristics of the tumor arerecognized. Since the invention results in amplification of pure tumorproduct, it becomes possible to characterize the tumor, in this caseusing sequence analysis. The assays methods of the invention thus permitan individual's tumor to be characterized without the need for biopsy orsurgery. Thus it becomes possible to treat tumors even before theybecome clinically evident, by starting treatment at latent stages,pre-recurrence stages, or even pre-malignant stages. Early treatment ofcancer before metastatic cells enter the bloodstream increases thelikelihood of cure.

Case 4

A 36 year old woman who has three small children has been diagnosed withbreast cancer two years ago. Her primary tumor had been shown tooverexpress a mutated c-myc oncogene. She had been treated with surgeryfollowed by a six month course of chemotherapy. In addition, her bloodserum has been evaluated for extracellular c-myc oncogene DNA using theassay methods of the invention. Specifically, extracellular DNA in serumis extracted using the silica extraction method, followed by c-mycspecific PCR amplification and ELISA detection of the c-myc specific PCRproducts. Although results for this patient are negative for some time,eventually her blood serum tests positive for extracellular c-myconcogene DNA using the methods of the invention. These results suggestan impending cancer recurrence. a multiplex panel of amplificationprimer pairs is used to analyze the patient's extracellular DNA fromserum, including primers specific for myc, ras, p53, EGFR, and HER-2/neuDNA, followed by sequencing. These data confirm that tumorcharacteristics are identical to those of the original primary breastcancer, confirming a recurrence of the patient's cancer rather than thedevelopment of a new primary tumor. Consequently, extracellular DNA inserum is measured quantitatively using a branched DNA signalamplification assay, with measurements performed 2 months and 4 monthslater. Quantitative measurements indicate increasing levels of c-mycDNA, and allow extrapolation to predict that clinical recurrence will benoted in approximately 2 years. This information allows both thephysician and the patient to plan future therapeutic options in thecontext of the patient's current social and family situation.

This hypothetical case illustrates the use of the invention to monitorpatients following therapy for recurrence of their cancer, to determinecharacteristics of their tumor, and to predict prognosis. Breast cancerpatients have a high incidence of second primaries, but the inventionpermits delineation of primary versus recurrent cancer by using amultiplex panel approach to evaluate tumor characteristics. Furthermore,since quantitative analysis permits clarification of prognosis, thepatient is in a better position to plan therapy within the context ofher social/family situation. Lastly, since the invention allowsdetection of tumor-derived extracellular DNA, and does not depend uponthe presence of circulating cancer cells, recurrence can be detected ata very early stage (in this hypothetical case, 2 years before clinicaldetection), which increases the likelihood of effective therapy.Effective therapy can also be planned based upon tumor characteristicssuggested by the extracellular DNA.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

1. A method of selecting a human with cancer for a cancer vaccinetherapy, the method comprising the steps of: a) extracting DNA fromblood plasma or serum of a human with cancer; b) amplifying or signalamplifying in a qualitative or quantitative fashion a portion of theextracted DNA using primers or probes specific to a DNA associated witha target of a cancer vaccine therapy; c) detecting the amplified orsignal amplified DNA, whereby a human with cancer is selected for thecancer vaccine therapy.
 2. The method of claim 1, wherein the target ofa cancer vaccine therapy is a protein associated with cancer.
 3. Themethod of claim 1, wherein the DNA is amplified in subpart (b) using anamplification method that is polymerase chain reaction, ligase chainreaction, boomerang DNA amplification, Q-beta replication,transcription-based amplification, isothermal nucleic acid sequencebased amplification, self-sustained sequence replication assay, stranddisplacement activation, or cycling probe technology.
 4. The method ofclaim 1, wherein the DNA is detected in subpart (c) using a detectionmethod that is gel electrophoresis, immunological detection, nucleicacid hybridization, Southern blot analysis, electrochemiluminescence,reverse dot blot hybridization, or high performance liquidchromatography.
 5. A method of selecting a human at risk for cancer orrecurrence of cancer for a cancer vaccine therapy, the method comprisingthe steps of: a) extracting DNA from blood plasma or serum of a human atrisk for cancer or recurrence of cancer; b) amplifying or signalamplifying in a qualitative or quantitative fashion a portion of theextracted DNA using primers or probes specific to a DNA associated witha target of a cancer vaccine therapy; c) detecting the amplified orsignal amplified DNA, whereby a human at risk for cancer or recurrenceof cancer is selected for the cancer vaccine therapy.
 6. The method ofclaim 5, wherein the target of a cancer vaccine therapy is a proteinassociated with cancer.
 7. The method of claim 5, wherein the DNA isamplified in subpart (b) using an amplification method that ispolymerase chain reaction, ligase chain reaction, boomerang DNAamplification, Q-beta replication, transcription-based amplification,isothermal nucleic acid sequence based amplification, self-sustainedsequence replication assay, strand displacement activation, or cyclingprobe technology.
 8. The method of claim 5, wherein the DNA is detectedin subpart (c) using a detection method that is gel electrophoresis,immunological detection, nucleic acid hybridization, Southern blotanalysis, electrochemiluminescence, reverse dot blot hybridization, orhigh performance liquid chromatography.
 9. A method of selecting a humanwith cancer for a cancer vaccine therapy, the method comprising thesteps of: a) extracting DNA from blood or a blood fraction of a humanwith cancer; b) amplifying or signal amplifying in a qualitative orquantitative fashion a portion of the extracted DNA using primers orprobes specific to a DNA associated with a target of a cancer vaccinetherapy; c) detecting the amplified or signal amplified DNA, whereby ahuman with cancer is selected for the cancer vaccine therapy.
 10. Amethod of selecting a human at risk for cancer or a cancer recurrencefor a cancer vaccine therapy, the method comprising the steps of: a)extracting DNA from blood or a blood fraction of a human at risk forcancer or recurrence of cancer; b) amplifying or signal amplifying in aqualitative or quantitative fashion a portion of the extracted DNA usingprimers or probes specific to a DNA associated with a target of a cancervaccine therapy; c) detecting the amplified or signal amplified DNA,whereby a human at risk for cancer or recurrence of cancer is selectedfor the cancer vaccine therapy.